Substrates and inhibitors of proteolytic enzymes

ABSTRACT

The present invention relates to the field of compounds which are substrates or inhibitors of proteolytic enzymes and to apparatus and methods for identifying substrates or inhibitors for proteolytic enzymes. We have devised a combinatorial method for the rapid identification of binding motifs which will greatly expedite the synthesis of inhibitors of a variety of proteolytic enzymes such as aspartyl proteases, serine proteases, metallo proteases and cysteinyl proteases.

[0001] The present invention relates to the field of compounds which aresubstrates or inhibitors of proteolytic enzymes and to apparatus andmethods for identifying substrates or inhibitors for proteolyticenzymes.

[0002] Many therapeutically useful drugs act as enzyme inhibitors. Inparticular, proteolytic enzyme inhibitors have been the focus of muchattention in the pharmaceutical industry, because they play a variety ofroles in a multitude of biological systems. Their proteolytic activitiesare related to processes ranging from cell invasion associated withmetastatic cancer to evasion of an immune response, as seen in certainis parasitic organisms; from nutrition to intracellular signalling tothe site-specific proteolysis of viral proteases and eukaryotichormone-processing enzymes. However, the traditional random screeningmethods for the identification of lead molecules as inhibitors ofproteolytic enzymes are often laborious and time-consuming. Thereforenew and efficient methods which can accelerate the drug discoveryprocess are greatly in demand.

[0003] We consider that proteases contain an active catalytic site whichtends to become increasingly activated as the recognition pockets¹ (S₁and S₂ etc) and (S₁′ and S₂′ etc) become better occupied. Therefore, itis important that those parts (P₁ and P₂ etc) (P₁′ and P₂′ etc) of theinhibitor that best fit into these pockets are identified as quickly aspossible in order to design novel protease inhibitors. Therefore, wehave devised a combinatorial method for the rapid identification ofthese binding motifs which will greatly expedite the synthesis ofinhibitors of a variety of proteolytic enzymes such as aspartylproteases, serine proteases, metallo proteases and cysteinyl proteases.

[0004] Proteases of interest include (but are not limited to):

[0005] 1. Aspartyl proteases, such as renin, HIV, cathepsin D andcathespin E etc.

[0006] 2. Metalloproteases, such as ECE, gelatinase A and B,collagenases, stromolysins etc.

[0007] 3. Cysteinyl proteases, such as apopain, ICI, DerPI, cathepsin B,cathepsin K etc.

[0008] 4. Serine proteases, such as thrombin, factor VIIa, factor Xa,elastase, trypsin.

[0009] 5. Threonyl proteases, such as proteasome S.

[0010] The use of a fluorescence resonance energy transfer (FRET)substrate for the analysis of proteolytic enzyme specificity was firstpublished by Carmel.² Since then many different quenched fluorogenicsubstrates for measuring enzyme inhibition have been described in theliterature.⁴⁻¹¹ These substrates contain a fluorophore, F, in a Pposition (vide supra), which is quenched by another group, Q, present ina P position (vide supra) and separated from F by the scissile bond. Theadvantage of the positioning of these residues, F and Q, is thatcleavage of a peptide bond occurs between the two natural residues and,therefore, represents a more natural hydrolytic event rather than thecleavage and release of a C-terminal chromophore.

[0011] For example, Bratovanova and Petkov¹² have synthesisedfluorogenic substrates from peptide 4-nitroanilides. N-acylation ofpeptide 4-nitroanilides with the aminobenzoyl (ABz) group yieldedsubstrates that are internally quenched by the presence of the4-nitroanilide moiety. Upon hydrolysis of the aminoacyl-4-nitroanilidebond, the highly fluorescent N-ABz group is released attached either toan amino acid or peptide.

[0012] Immobilised libraries, where substrates are attached to a polymeror biopolymer support, have also been used for mapping protease bindingsites.¹³ Singh et al. reported recently that enzymatic substrateactivity of 38 selected octapeptides attached via a linker to controlledpore glass is predictive of the same activity of similar peptides insolution. However, these results are preliminary and only for a specificexample. Therefore, it is not clear whether immobilised substratesattached to polymers can reliably replace soluble substrates in mappingthe hindered protease binding sites, especially since the hydrophilic orlipophilic nature of the polymer and the size of the interstices withinthe polymer are bound to influence the reaction between the enzyme andits substrates.

[0013] Mixtures of internally quenched, fluorogenic substrates have alsorecently been described in which the quencher group, Q, is2,4-dinitrophenyl (Dnp) and is attached to the P side of the scissilebond, while the fluorogenic group, is N-methyl anthranilic acid (Nma)and is attached to the P′ side.¹⁴

[0014] Examples of other Donor-Acceptor Chromophore Pairs that have beenapplied to Biological Systems are shown in Table 1. TABLE 1Donor-Acceptor Chromophore Pairs That Have Been Applied To BiologicalSamples Donor Acceptor Naphthalene Dansyl IANBD DDPM IAEDANS DDPM DNSMLY IAEDANS IANBD E-A F₂DNB Pyrene Bimane ANAI IPM IAANS IAF ε-A F₂DPSε-A DDPM IAEDANS TNP MNA DACM PM NBD FITC TNP-ATP DANZ DABM NCP CPM NAADNP LY TNP-ATP IAF diI—C₁₈ IAF TMR FMA FMA PM DMAMS mBBR FITC mBBR DABMε-A NBD Pyrene Coumarin IPM FNAI IAEDANS DABM IAEDANS TNP-ATP ε-A IANBDNBD SRH ISA TNP Dansyl ODR DANZ IAF FNAI EITC NBD LRH IAF EIA FITC ENAIProflavin ETSC CPM TNP-ATP IAEDANS IAF CPM Fluorescein IAEDANS FITC FITCTMR IAF TMR CF TR CPM FTS ε-A TNP-ATP CPM FM LY EM FITC EITC IAEDANSDiO—C₁₄ IAF ErITC FITC EM FITC ETSC FITC ErITC BPE CY5

[0015] ANAI, 2-anthracene N-acetylimidazole; BPE, B-phycoerythrin; CF,carboxyfluorescein succinimidyl ester; CPM,7-doethylamino-3-(4′maleimidylphenyl)-4-methylcoumarin; CY5,carboxymethylindocyanine-N-hydroxysuccinimidyl ester, diI-C₁₈,1,1′-dioctadecyl-3,3,3′3′-tetramethyl-indocarbocyanine; DiO-C₁₄,3,3′-ditetradecyloxacarbocyanine; DABM,4-dimethylaniniphenylazo-phenyl-4′-maleimide; DACM,(7-(dimethylamino)coumarin-4-yl)-acetyl; DANZ, dansylaziridine; DDPM,N-(4-dimethylamino-3-5-dinitrophenyl)maleimide; DACM,di-methylamino-4-maleimidostilbene; DMSM, N-(2,5-dimethoxystiben-4-yl)maleimide; DNP, 2,4-dinitrophenyl; ε-A, I,N⁶-ethenoadenosine; EIA,5-(iodoacetetamido)eosin; EITC, eosin-5-isothiocyanate; ENAI, eosinN-acetylimidazole; EM, eosin maleimide; ErITC,erythrosin-5′-isothiocyanate; ETSC, eosin thiosemicarbazide; F₂DPB,1,5-difluoro-2,4′dinitrobenzene; F₂DPS,4,4′-difluro-3,3′dinitropheylsulphone; FITC, fluoresceinN-acetylimidazole; FTS, fluorescein thiosemicarbazide; IAANS;2-((4′-iodoacetamido)anilino)naphthalene-6-sulphonic acid; IAEDANS,5-(2-((iodoacetyl)amino)ethylamino)-naphthlene-1-sulphonic acid; IAF,5-iodoacetamidofluorescein; IANBD,N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa1,3,diazole;IPM, 3(4-isothiocyanatophenyl)7-diethyl-4-methylcoumarin; ISA,4-(iodoacetamido)salicylic acid; LRH,lissaminerho-2,1,3-benzoxadiazol-4-yl; NCP,N-cyclohexyl-N′-(1-pyrenyl)carbodiimide; ODR, octadecylrhodamine; PM,N-(1-pyrene)-maleimide; SRH sulphurhodamine; TMR, tetramethylrhodamine;TNP, trinitrophenyl; TR, Texas red.

[0016] from: Wu, P. and Brand, L. 1994. Anal. Biochem. 218, 1-13.

[0017] The specificity of soluble peptide libraries have beendetermined.¹⁵⁻¹⁶ Berman et al. described¹⁶ an HPLC mass spectrometrytechnique in which 6 mixtures of 128 peptides were synthesised whichwere N-terminally labelled with the Dnp group in order to allow UVmonitoring on the HPLC. The disadvantage of this approach is that eachassay mixture has to be individually analysed, because no fluorogenicsubstrate is revealed, and that the effective concentration of eachseparate component is limited by the size of the mixture because ofoverall solubility factors. Drevin et al.¹⁷ have suggested the use ofindividually synthesised fluorogenic substrates for the determination ofenzyme activity using a chromophore which chelates lanthamide ions.Garmann and Phillips have suggested the use of FRET substrates in whichthe fluorogenic and quencher moieties are attached via thiol or aminofunctional groups after the peptide has been synthesised, but this hasthe disadvantage that they are not in library form and that thesefunctional amino and thiol groups need to be selectively revealed afterthe peptide has been synthesised. Wang et al. have suggested the use ofthe EDANS and DABCYL fluorescor and quencher pairing for the individualsynthesis of substrates for proteolytic enzymes.

[0018] The above methods which have used FRET techniques for the mappingof the active site around a specific protease suffer from one or more ofthe following disadvantages:

[0019] i. because of general aqueous insolubility they do not producemixtures of compounds in a form suitable for high throughput screeningin aqueous solution.

[0020] ii. the derivatised compounds cannot be prepared in combinatoriallibrary form using solid phase techniques.

[0021] iii. the mixtures which have been used^(8,9) were notself-decoding, and needed time-consuming deconvolutive resynthesis foridentification of the active molecules.

DESCRIPTION OF THE DRAWINGS

[0022] FIGS. 1 to 14 inclusive exemplify component distributions in theplates of a library matrix;

[0023]FIG. 15 illustrates a reaction scheme for production of compoundnumber 4: Boc-Val-Ala-Leu-H wherein

[0024] i. isobutylchloroformate, N-methylmorpholine, thenN,O-dimethylhydroxylamine HCl, THF.

[0025] ii. HCl, dioxan. iii. isobutylchloroformate, N-methylmorphline,then Soc-Ala-OH, THF.

[0026] iv. HCl, dioxan. V. Boc-Val-Osuc, N-methylmorpholine, DMF. Vi.LAH;

[0027]FIG. 16 illustrates a reaction scheme for production of activeinhibitors of Der P1.

BRIEF DESCRIPTIONS OF THE INVENTION

[0028] The present invention relates to the field of:

[0029] i. Compounds which are substrates or inhibitors of proteolyticenzymes.

[0030] ii. Apparatus and methods which provide the rapid generation ofstructure-activity relationships using auto-deconvoluting combinatoriallibraries, which facilitate the invention of novel inhibitors ofproteolytic enzymes.

[0031] iii. Apparatus and methods which provide the detection andmeasurement of proteolytic enzyme activity using combinatorial FRET(fluorescence resonance energy transfer) libraries of molecules.

[0032] iv. Apparatus and methods which provide the establishment ofbiological assays for proteolytic enzymes through the rapid discovery ofhighly active substrates for proteolytic enzymes.

[0033] We describe herein apparatus and methods which can be used forthe rapid generation of structure-activity relationship (SAR) data and,therefore, the characterisation of the binding motif of any protease,and which will, therefore, facilitate:

[0034] i. the development of a sensitive enzyme inhibition assay byusing the best compound in the library as the fluorogenic substrate forthe proteolytic enzyme under scrutiny.

[0035] ii. the invention of novel compounds which are proteolytic enzymeinhibitors by rapid characterisation of the best binding motif.

[0036] iii. computer aided drug design to design potent inhibitors usingknown methodology, and also in prioritising which pre-synthesisedcompounds in the in-house and commercially available databases to assay.

[0037] In a first aspect the invention provides novel compoundsrepresented by the formula A-B-C-D-nE-F [I] in which;

[0038] A represents a fluorescor internally quenched by F;

[0039] B, C, D, and E represent groups such that the scissile bondbetween any two of these groups is a suitable bond;

[0040] F represents a quencher capable of internally quenching thefluorescor A; and

[0041] n represents an integer between 1 and 4 inclusive.

[0042] In some embodiments the suitable bond is an unsubstituted amidebond (see Example 1); in other embodiments the suitable bond is an esterbond (see Example 2).

[0043] In preferred embodiments B, C, D, E are amino acids or hydroxyacids. That is a molecule with an amine or hydroxy terminus and acarboxylic acid terminus. The amine/hydroxy group may be positioned onthe same carbon atom or separated by a number of atoms and atom types.

[0044] The molecules may be cyclic or alicyclic. They may be linear orcyclic on the same structure

[0045] or BCDE can be on a central scaffold (linear or cyclic)

[0046] or one of the residues can be the backbone

[0047] Where the symbol -B-C-D-E- or -B-C-D-nE- is used herein, it is tobe understood to include all of these linear and cyclic variants withinits definition.

[0048] At least one but not all the bonds between B, C, D, E need to bescissile. Non scissile bonds may include sulphonamide, urea,aminomethylene.

[0049] Several non-limiting examples of “scaffold” molecules are shownin Table 2, in which substituents R₁-R₄ correspond to possible variablegroups B, C, D and E.

[0050] In a second aspect the invention provides a combinatorial libraryof FRET compounds comprising a mixture of compounds of formula [I].

[0051] In a third aspect the invention provides for the use of such acombinatorial FRET library in a method which provides rapid generationof structure-activity relationships (SAR) which comprises detection andmeasurement of proteolytic enzyme activity by carrying out an assay witha library of combinatorial FRET (fluorescence resonance energy transfer)molecules to find a substrate or substrates for the enzyme. According tothis method an identified substrate can be synthesised and used inbiological assay for proteolytic enzymes. Novel substrates are includedin the scope of the invention.

[0052] In a forth aspect the invention provides for the use of such acombinatorial FRET library in a method for detection and measurement ofproteolytic enzyme activity against compounds of the library.

[0053] In a fifth aspect the invention provides a method which comprisesthe identification of an enzyme inhibitor or inhibitors wherein a FRETcompound which has been

[0054] identified as a substrate is used in an inhibition assay with theenzyme separately against a panel of possible inhibitors.

[0055] In a sixth aspect the invention provides a set of compounds whichcomprises two complementary FRET compound libraries. Such a set will bereferred to hereafter as “apparatus” because it allows for the screeningor assay method for identifying substrates or inhibitors of proteolyticenzymes. This set of compounds constituting an apparatus is capable ofproviding an auto-deconvoluting combinatorial library as will bedescribed below.

[0056] In a seventh aspect the invention provides a method ofidentifying and synthesising an inhibitor of a proteolytic enzyme whichcomprises detection and measurement of proteolytic enzyme activity bycarrying out an assay with a library of combinatorial FRET (fluorescenceresonance energy transfer) molecules, deconvoluting the library to finda substrate or substrates for the enzyme and synthesis of an inhibitorbased on the substrate or substrates. The direct product of this methodis one or more novel proteolytic enzyme inhibitors.

[0057] In an eighth aspect the invention provides an inhibition assaywhich uses a FRET molecule, which has been identified as a substrate forthe enzyme, wherein the molecule is assayed with the enzyme separatelyagainst a panel of possible inhibitors.

[0058] In a ninth aspect the invention provides a complementary pair ofcompound libraries L1 and L2 which constitute a set containing compoundsof formula:

Aa-Bb-Cc-Dd-n(Ee)-Ff-Gg

[0059] giving a×b×c×d×e×f×g=Mn compounds in each library, there being apredetermined number (P1, P2) of mixtures each consisting of apredetermined number (Q1, Q2) of individual identifiable compounds ineach library, wherein both L1 and L2 contain the same Mn compounds, butwherein any two compounds which are found together in one mixture of Q1compounds of L1 are not found together in any one of the P2 mixtures ofL2.

[0060] In a tenth aspect the invention provides a method of screeningfor enzymic activity using the libraries L1, L2 is described above inwhich the P1 mixtures of L1 and the P2 mixtures of L2 are each placedseparately into individual wells of well plates, the well plates havingwells arranged in a format adapted to allow deduction of a unique activecompound formula from the presence of activity in one well of L1 and onewell of L2.

[0061] The apparatus of the invention preferably comprises twocomplementary compound libraries, L1 and L2, each containing n×1600compounds of the invention, of the type A-B-₁₋₁₀-C₁₋₁₀-D₁₋₈-n(E₁₋₂)-F-G[II], in which:

[0062] A=a fluorescor internally quenched by F, preferably anunsubstituted or substituted anthranilic acid derivative, connected byan amide bond to B

[0063] B, C, D, E, are natural or unnatural amino acid residuesconnected together by suitable bonds, although B, C, D and E can be anyset of groups, provided that the scissile bond between D-E is anunsubstituted bond.

[0064] F=a quencher capable of internally quenching the fluorescor A,preferably an unsubstituted or substituted 3-nitrotyrosine derivative.

[0065] G=optionally present and is a hydrophilic moiety, preferably anaspartyl amide moiety. If present, G advantageously ensures that allcompounds in the library are imparted with aqueous solubility. Also, Gshould not be a substrate for any type of enzyme.

[0066] n=any integer between 1 and 4 inclusive.

[0067] In an alternative, the scissile bond could be between B-C is orC-D.

[0068] (Note that A and F herein correspond generally and respectivelyto moieties F and Q of the prior art referred to above).

[0069] The numbers represented in subscript following residues B, C, Dand E refer to the number of possibilities from which those residues areselected. Thus, by way of illustrative example, A-B₁₋₅-C-D-E₁₋₂-F-Grepresents a mixture of the following ten compounds:

[0070] A-B₁-C-D-E₁-F-G

[0071] A-B₂-C-D-E₁-F-G

[0072] A-B₁-C-D-E₁-F-G

[0073] A-B₄-C-D-E₁-F-G

[0074] A-B₁-C-D-E₁-F-G

[0075] A-B₁-C-D-E₂-F-G

[0076] A-B₁-C-D-E₂-F-G

[0077] A-B₁-C-D-E₂-F-G

[0078] A-B₁-C-D-E₂-F-G

[0079] A-B₁-C-D-E₂-F-G

[0080] The general combinatorial formula for each library can beexpressed as:

A₁-B₁₀-C₁₀-D₈-n(E₂)-F₁-G₁  [III]

[0081] providing 1×10×10×8×n×2×1×1=1600n compounds.

[0082] Both compound libraries, L1 and L2, of the above type aresynthesized using solid phase techniques using the Multipin approach²⁴such that each library contains 1600n compounds as 80n mixtures of 20distinct, identifiable compounds. These 20 component mixtures are thenplaced separately into each of 80 wells of a 96 well plate (the othertwo lanes are used for control experiments) and then screened against aknown quantity of the protease.

[0083] Thus it is an important part of the invention that regardless ofthe number of compounds contained in the two libraries L1 and L2 (e.g.in the preferred embodiment: 1600n, where n=any integer between 1 and 4)the libraries themselves are complementary and amenable to deconvolutionwithout recourse to resynthesis. It is also an important part of theinvention that the library matrix has been especially formatted so thatthe most important site pairings P₂ and P₁ for proteolytic enzymes canbe identified immediately without recourse to resynthesis. Thosecompounds of the type A-B-C-D-E-F-G that are the better substrates forthe protease will be cleaved, and can be readily identified because thefluorescor, A, will be cleaved from its nearby quencher F, in a timedependent manner which can be easily quantified. The fluorescentquenching by F of A only occurs when the two are in nearby proximity,normally within 30 angstrom units. Hence cleavage of a scissile bond(e.g. the scissile bond D-E) allows F to move further away from A andthus allow A to fluoresce when excited by light of the correctwavelength.

[0084] 1. In this manner the most active compound can be rapidlyidentified without the need for further resynthesis and deconvolution.Moreover, the wells that show the most rapid development of fluorescencecan also be analysed by mass spectrometry, since by comparison with theoriginal mixture, the identity of the most efficient substrate can befound by its disappearance into its two component parts, e.g A-B-C-D andE-F-G.

[0085] Hence the problem of library deconvolution can be overcome andthe most active substrate for the enzyme can be rapidly identified.

[0086] In addition, after the initial treatment of the proteolyticenzyme with the library mixtures, L1 and L2, the residual enzymaticactivity in each well can be quantified by the addition of the mostpotent fluorogenic substrate for the enzyme, S1, which is found in the16×n compound library. Because of the nature of the library design thiscan be quickly prepared and purified. If there is no appearance ofincreased fluorescence with the known substrate, S1, then the presenceof an enzyme inhibitor can be inferred, which again can be quicklyidentified without the need for resynthesis.

[0087] The general description of the library layout will now bedescribed with reference to FIGS. 1 to 14.

[0088] For example, when n=1 and the library contains 1600 compounds, inthe first column of the first row (A1) (FIG. 1) in the first plate (P1)of the library L1, (hereinafter designated as location A1,P1,L1) therewill be one C component, C₁, one D component, D₁, the ten B componentsand the two E components (E₁ and E₂) (FIG. 2). In the tenth column ofthe first row (A10) in the first plate (P1) of the library L1,(hereinafter designated as location A10,P1,L1) there will be one Ccomponent, C₁₀, one D component, D₁, the ten B components and the two Ecomponents (E₁ and E₂). In the tenth column of the eighth row (H10) inthe first plate (P1) of the library L1, (hereinafter designated aslocation H10,P1,L1) there will be one C component, C₁₀, one D component,D₈, the ten B components and the two E components (E₁ and E₂). Hence all1600 components are present in the one plate, because the 80 wells eachcontain 20 components.

[0089] A second complementary library is synthesised as follows (FIG.3). In the first column of the first row (A1) of the first plate (P1) ofthe library, L2, (hereinafter designated as location A1,P1,L2), therewill be ten C components, two D components (D₁ and D₂), one B component,B₁, and one E component, E₁. In the tenth column of the first row (A10)of the first plate (P1) of the library, L2, (hereinafter designated aslocation A10,P1,L2), there will be ten C components, two D components(D₁ and D₂), one B component, B₁₀, and one E component, E₁. In the firstcolumn of the second row (B1) of the first plate (P1) of the library,L2, (hereinafter designated as location B1,P1,L2), there will be ten Ccomponents, two D components (D₁ and D₂), one B component, B₁, and one Ecomponent, E₂. In the tenth column of the second row (B10) of the firstplate (P1) of the library, L2, (B10,P1,L2) there will be ten Ccomponents, two D components (D₁ and D₂), one B component, B₁₀, and oneE component, E₂. Hence only the first two rows are used to accommodate400 compounds in total.

[0090] In the first column of the first row (A1) of the second plate(P2) of the library, L2, (hereinafter designated as location A1,P2,L2),there will be ten C components, two D components (D₃ and D₄), one Bcomponent, B₁, and one E component, E₁ (FIG. 4). In the tenth column ofthe first row (A10) of the second plate (P2) of the library, L2,(hereinafter designated as location A10,P2,L2), there will be ten Ccomponents, two D components (D₃ and D₄), one B component, B₁₀, and oneE component, E₁. In the first column of the second row (B1) of thesecond plate (P2) of the library, L2, (hereinafter designated aslocation B1,P2,L2), there will be ten C components, two D components (D₃and D₄), one B component, B₁, and one E component, E₂. In the tenthcolumn of the second row (B10) of the second plate (P2) of the library,L2, (B10,P2,L2), there will be ten C components, two D components (D,and D₄), one B component, B₁₀, and one E component, E₂. Hence only thefirst two rows are used to accommodate 400 compounds in-total.

[0091] In the first column of the first row (A1) of the third plate (P3)of the library, L2, (hereinafter designated as location A1,P3,L2), therewill be ten C components, two D components (D₅ and D₆), one B component,B₁, and one E component, E1 (FIG. 5). In the tenth column of the firstrow (A10) of the third plate (P3) of the library, L2, (hereinafterdesignated as location A10,P3,L2), there will be ten C components, two Dcomponents (D₅ and D₆), one B component, B₁₀, and one E component, E₁.In the first column of the second row (B1) of the third plate (P3) ofthe library, L2, (hereinafter designated as location B1,P3,L2), therewill be ten C components, two D components (D₅ and D₆), one B component,B₁, and one E component, E₂. In the tenth column of the second row (B10)of the third plate (P3) of the library, L2, (B10,P3,L2), there will beten C components, two D components (D₅ and D₆), one B component, B₁₀,and one E component, E₂. Hence only the first two rows are used toaccommodate 400 compounds in total.

[0092] In the first column of the first row (A1) of the fourth plate(P4) of the library, L2, (hereinafter designated as location A1,P4,L2),there will be ten C components, two D components (D₇ and D₈), one Bcomponent, B₁, and one E component, E₂ (FIG. 6). In the tenth column ofthe first row (A10) of the fourth plate (P4) of the library, L2,(hereinafter designated as location A10,P4,L2), there will be ten Ccomponents, two D components (D₇ and D₈), one B component, B₁₀, and oneE component, E₁. In the first column of the second row (B1) of thefourth plate (P4) of the library, L2, (hereinafter designated aslocation B1,P4,L2), there will be ten C components, two D components (D₇and D₈), one B component, B₁, and one E component, E₂. In the tenthcolumn of the second row (B10) of the fourth plate (P4) of the library,L2, (B10,P4,L2), there will be ten C components, two D components (D₇and D₈), one B component, B₁₀, and one E component, E₂. Hence only thefirst two rows are used to accommodate 400 compounds in total.

[0093] In this fashion two complementary libraries, L1 and L2 areprepared. In library, L1, each of the 80 of wells contains a mixture of20 components providing 1600 compounds for screening. In library, L2,four plates are used in which only the first two rows are employed,providing 20 wells of 20 components per well per plate, and furnishingthe same 1600 compounds as are present in library L1, but in a format inwhich no two compounds found together in library, L1, will be foundtogether in library, L2.

[0094] Thus it is an important part of the invention that the compoundscontained in the two libraries L1 and L2 are themselves complementary,in that any two compounds which are found together in a 20 componentmixture in the same location (e.g. A1P1L1) in library L1, are not foundtogether in any of the 20 component mixtures in any location of thelibrary L2.

[0095] Thus, for example, with reference to the primary library P1 L1 ofFIG. 2 and the secondary libraries P1 L2, P2 L2, P3 L2 and P4 L2 ofFIGS. 3-6 it is possible to deconvolute an examplary sequence:

[0096] -B₂-C₃-D₄-E₁-

[0097] If this sequence is a substrate fluorescence will occur in P1 L1at C₃D₄. This gives the information that the substrate is

[0098] -?-C₃-D₄-?-

[0099] If fluorescence occurs in P2 L2 at B₂E₁ it indicates a substrate

[0100] -B₂-?-?-E₁-

[0101] The confirmation of the substrate as

[0102] -B₂-C₃-D₄-E₁-

[0103] should be provided by non-fluorescence of P1 L2, P3 L2 and P4 L2which all contain -B₂-C₃-X-E₁- where X is not D₄.

[0104] In practice it is likely that more than one sequence will resultin a substrate. Information as to which positions B-C-D-E- are sensitiveto change (i.e. require a specific group) and which are insensitive(i.e. can tolerate more than one choice of group) in the context of thewhole sequence gives valuable SAR data which can be used to model and/orsynthesise related compounds.

[0105] In analogous examples, where separately n=2, 3 or 4, extra platesare constructed in library L1 format to accommodate the component pairsE₃ and E₄ (n=2), E₅ and E₆ (n=3), and E₇ and E₈ (n=4), respectively. Forthe respective deconvolution libraries of the type, L2, the respectiverows in the plates P1, P2, P3, and P4, are increasingly filled with thepaired components D₁ and D₂, D₃ and D₄, and D₅ and D₆, and D₇ and D₈,respectively.

[0106] For example, when n=3, and the library contains 4800 compounds,in the first column of the first row (A1) in the first plate (P1) of thelibrary L1, (hereinafter designated as location A1,P1,L1) there will beone C component, C₁, one D component, D₁, the ten B components and thetwo E components (E₁ and E₂). In the tenth column of the first row (A10)in the first plate (P1) of the library L1, (hereinafter designated aslocation A10,P1,L1) there will be one C component, C₁₀, one D component,D₁, the ten B components and the two E components (E₁ and E₂) In thetenth column of the eighth row (H10) in the first plate (P1) of thelibrary L1, (hereinafter designated as location H10,P1,L1) there will beone C component, C₁₀, one D component, D₈, the ten B components and thetwo E components (E₁ and E₂). Hence 1600 components are present in theone plate, because the 80 wells each contain 20 components.

[0107] In the first column of the first row (A1) in the second plate(P2) of the library L1, (hereinafter designated as location A1,P2,L1)there will be one C component, C₁, one D component, D₁, the ten Bcomponents and the two E components (E₃ and E₄). In the tenth column ofthe first row (A10) in the second plate (P2) of the library L1,(hereinafter designated as location A10,P2,L1) there will be one Ccomponent, C₁₀, one D component, D₁, the ten B components and the two Ecomponents (E₃ and E₄). In the tenth column of the eighth row (H10) inthe second plate (P2) of the library L1, (hereinafter designated aslocation H10,P1,L1) there will be one C component, C₁₀, one D component,D₈, the ten B components and the two E components (E₃ and E₄). Hence1600 components are present in the one plate, because the 80 wells eachcontain 20 components.

[0108] In the first column of the first row (A1) in the third plate (P3)of the library L1, (hereinafter designated as location A1,P3,L1) therewill be one C component, C₁, one is D component, D₁, the ten Bcomponents and the two E components (E₅ and E₆). In the tenth column ofthe first row (A10) in the third plate (P3) of the library L1,(hereinafter designated as location A10,P3,L1) there will be one Ccomponent, C₁₀, one D component, D₁, the ten B components and the two Ecomponents (E₅ and E₆). In the tenth column of the eighth row (H10) inthe third plate (P3) of the library L1, (hereinafter designated aslocation H10,P3,L1) there will be one C component, C₁₀, one C component,C₈, the ten B components and the two E components (E₅ and E₆). Hence1600 components are present in the one plate, because the 80 wells eachcontain 20 components. In total the three plate, P1, P2 and P3, contain1600 compounds/plate 4800 compounds in total.

[0109] For example, when n=4, and the library contains 6400 compounds,in the first column of the first row (A1) in the first plate (P1) of thelibrary L1, (hereinafter designated as location A1,P1,L1) there will beone C component, C₁, one D component, D₁, the ten B components and thetwo E components (E₁ and E₂) (FIG. 7). In the tenth column of the firstrow (A10) in the first plate (P1) of the library L1, (hereinafterdesignated as location A10,P1,L1) there will be one C component, C₁₀,one D component, D₁, the ten B components and the two E components (E₁and E₂). In the tenth column of the eighth row (H10) in the first plate(P1) of the library L1, (hereinafter designated as location H10,P1,L1)there will be one C component, C₁₀, one D component, D₈, the ten Bcomponents and the two E components (E₁ and E₂). Hence all 1600components are present in the one plate, because the 80 wells eachcontain 20 components.

[0110] In the first column of the first row (A1) in the second plate(P2) of the library L1, (hereinafter designated as location A1,P2,L1)there will be one C component, C₁, one D component, D₁, the ten Bcomponents and the two E components (E₃ and E₄) (FIG. 8). In the tenthcolumn of the first row (A10) in the second plate (P2) of the libraryL1, (hereinafter designated as location A10,P2,L1) there will be one Ccomponent, C₁₀, one D component, D₁, the ten B components and the two Ecomponents (E₃ and E₄). In the tenth column of the eighth row (H10) inthe second plate (P2) of the library L1, (hereinafter designated aslocation H10,P2,L1) there will be one C component, C₁₀, one D component,D₈, the ten B components and the two E components (E₃ and E₄).

[0111] In the first column of the first row (A1) in the third plate (P3)of the library L1, (hereinafter designated as location A1,P3,L1) therewill be one C component, C₁, one D component, D₁, the ten B componentsand the two E components (E₅ and E₆) (FIG. 9). In the tenth column ofthe first row (A10) in the third plate (P3) of the library L1,(hereinafter designated as location A10,P3,L1) there will be one Ccomponent, C₁₀, one D component, D₁, the ten B components and the two Ecomponents (E₅ and E₆). In the tenth column of the eighth row (H10) inthe third plate (P3) of the library L1, (hereinafter designated aslocation H10,P3,L1) there will be one C component, C₁₀, one D component,D₈, the ten B components and the two E components (E₅ and E₆).

[0112] In the first column of the first row (A1) in the fourth plate(P4) of the library L1, (hereinafter designated as location A1,P4,L1)there will be one C component, C₁, one D component, D₁, the ten Bcomponents and the two E components (E₇ and E₈) (FIG. 10). Likewise, inthe tenth column of the first row (A10) in the fourth plate (P4) of thelibrary L1, (hereinafter designated as location A10,P4,L1) there will beone C component, C₁₀, one D component, D₁, the ten B components and thetwo E components (E₇ and E₈). In the tenth column of the eighth row(H10) in the fourth plate (P4) of the library L1, (hereinafterdesignated as location H10,P4,L1) there will be one C component, C₁₀,one D component, D₈, the ten B components and the two E components (E₇and E₈)

[0113] A second complementary library is synthesised as follows. In thefirst column of the first row (A1) of the first plate (P1) of thelibrary, L2, (hereinafter designated as location A1,P1,L2), there willbe ten C components, two D components (D₁ and D₂), one B component, B₁,and one E component, E₁ (FIG. 11). In the tenth column of the first row(A10) of the first plate (P1) of the library, L2, (hereinafterdesignated as location A10,P1,L2), there will be the ten C components,two D components (D₁ and D₂), one B component, B₁₀, and one E component,E₁. In the first column of the eighth row (H1) of the first plate (P1)of the library, L2, (hereinafter designated as location H1,P1,L2), therewill be the ten C components, two D components (D₁ and D₂), one Bcomponent, B₁, and one E component, E₈. In the tenth column of theeighth row (H10) of the first plate (P1) of the library, L2, (H10,P1,L2)there will be the ten C components, two D components (D₁ and D₂), one Bcomponent, B₁₀, and one E component, E₈. Hence the matrix containing allten columns and all eight rows are used to accommodate 1600 compounds intotal.

[0114] In the first column of the first row (A1) of the second plate(P2) of the library, L2, (hereinafter designated as location A1,P2,L2),there will be ten C components, two D components (D₃ and D₄), one Bcomponent, B₁, and one E component, E₁ (FIG. 12). In the tenth column ofthe first row (A10) of the second plate (P2) of the library, L2,(hereinafter designated as location A10,P2,L2), there will be ten Ccomponents, two D components (D₃ and D₄), one B component, B₁₀, and oneE component, E₁. In the first column of the second row (B1) of thesecond plate (P2) of the library, L2, (hereinafter designated aslocation B1,P2,L2), there will be ten C components, two D components (D₃and D₄), one B component, B₁, and one E component, E₂. In the tenthcolumn of the eighth row (H10) of the second plate (P2) of the library,L2, (H10,P2,L2), there will be ten C components, two D components (D₃and D₄), one B component, B₁₀, and one E component, E₈.

[0115] In the first column of the first row (A1) of the third plate (P3)of the library, L2, (hereinafter designated as location A1,P3,L2), therewill be ten C components, two D components (D₅ and D₆), one B component,B₁, and one E component, E₁ (FIG. 13). In the tenth column of the firstrow (A10) of the third plate (P3) of the library, L2, (hereinafterdesignated as location A10,P3,L2), there will be ten C components, two Dcomponents (D₅ and D₆), one B component, B₁₀, and one E component, E₁.In the first column of the second row (B1) of the third plate (P3) ofthe library, L2, (hereinafter designated as location B1,P3,L2), therewill be ten C components, two D components (D₅ and D₆), one B component,B₁, and one E component, E₂. In the tenth column of the eighth row (H10)of the third plate (P3) of the library, L2, (H10,P3,L2), there will beten C components, two D components (D₅ and D₆), one B component, B₁₀,and one E component, E₈.

[0116] In the first column of the first row (A1) of the fourth plate(P4) of the library, L2, (hereinafter designated as location A1,P4,L2),there will be ten C components, two D components (D₇ and D₈), one Bcomponent, B₁, and one E component, E₁ (FIG. 14). In the tenth column ofthe first row (A10) of the fourth plate (P4) of the library, L2,(hereinafter designated as location A10,P4,L2), there will be ten Ccomponents, two D components (D₇ and D₈), one B component, B₁₀, and oneE component, E₁. In the first column of the second row (B1) of thefourth plate (P4) of the library, L2, (hereinafter designated aslocation B1,P4,L2), there will be ten C components, two D components (D₇and D₈), one B component, B₁, and one E component, E₂. In the tenthcolumn of the eighth row (H10) of the fourth plate (P4) of the library,L2, (H10,P4,L2), there will be ten C components, two D components (D₇and D₈), one B component, B₁₀, and one E component, E₈.

[0117] It will be apparent to those skilled in the art that thesynthesis of two orthogonal sets of mixtures in solution providing twocomplementary FRET libraries indexed in two dimensions forautodeconvolution does not require the currently preferred arrangement-B₁₋₁₀-C₁₋₁₀-D₁₋₈-E₁₋₈-.

[0118] The general concept of two orthogonal sets of mixtures indexed intwo dimensions can be applied to various permutations of numbers ofwells, plate layout, number of permutations per mixture etc.

[0119] A numerical interrelationship can be defined as follows:

[0120] General Deconvolution Formulae

-Bb-Cc-Dd-n(Ee)-  (I)

[0121] 1) Primary and Secondary plates preferably have the same numberof compounds per well [X]: otherwise there are two values, having X_(p)and X_(s) respectively.

[0122] 2) The primary library comprises [np] plates.

[0123] If Rp.Cp=Rs.Cs, then the number of plates in the secondarylibrary is also [np]. If not, the number of plates in the secondarylibrary [ns] is:${n\quad s} = {\frac{{Rp} \cdot {Cp}}{{Rs} \cdot {Cs}} \cdot {np}}$

[0124] eg. A primary library of np=4, Rp=8, Cp=10 can be set out in anRs=4, Cs=S secondary library with the number of plates equal to:$\begin{matrix}{{:{n\quad s}} = \quad {\frac{8 \times 10}{4 \times 5} \cdot {np}}} \\{= \quad {16\quad {{plates}.}}}\end{matrix}$

[0125] Number of compounds per well

-Bb-Cc-Dd-np(Ee)-  (1)

[0126] Number of possible combinations [k] is given by:

k=b.c.d.np.e  (2)

[0127] When number of wells on a plate=[N], number of compounds perwell=[α]and number of plates=[np]:

k=X.N.np  (3)

[0128] However, number of wells [N] is also defined by the number ofrows [Rp] and number of columns [Cp]:

N=Rp.Cp  (4)

[0129] Combining (3) and (4):

k=X.Rp.Cp.np  (5)

[0130] Combining (2) and (5):

b.c.d.np.e=X.Rp.Cp.np  (6)

[0131] Cancelling [np] from both sides of the equation:

b.c.d.e=X.Rp.Cp  (7)

[0132] Two of the variables (e.g. b.c) on the left side of the equationmust each be equal in number to the number of columns [Cp], whilst aremaining variable (e.g. d) on the left side must be equal in number tothe number of rows [Rp]. So:

[Cp]₂.Rp.e=X.Rp.Cp  (8)

[0133] Cancelling [Cp] and [Rp] from both sides of the equation:

Cp.e=X  (9)

[0134] where [e] is the number of variants along a fixed row; and

[0135] if Rp=Cp, then Rp.e=X.

EXAMPLE

[0136] for a 10×10×8×8 format over 4 plates:

[0137] np.e=8=>e=2

[0138] 10×2=X

[0139] X=20.

[0140] From an understanding of the general deconvolution formulae shownabove, those skilled in the art will readily appreciate that theadvantageous results of self-deconvolution according to the inventionare obtainable utilising a number of different arrangements of wells,plate layouts, mixtures etc and that such variants on the preferredembodiment illustrated herein are intended to be within the scope of thepresent invention.

[0141] The FRET strategy is based on the synthesis of two orthogonalsets of mixtures in solution. These solutions are each indexed in twodimensions. Thus the data from, for example, a protease scan identifiesthe most active compounds without the need for decoding or resynthesis.The positional preferences of sub-units (in this case amino acids) areoptimised with respect to all other variant positions simultaneously.The synergistic relationship between all four positions is realised andboth positive, beneficial and negative, deactivating data are generated.This leads to families (sub-populations) of substrates and theirsub-unit preferences. The data can be fed into molecular modellingprograms to generate pharmacophoric descriptors that encompass both thedesirable features (from the positive data) and indicate undesirableinteractions (from the negative data sets). Note that a one dimensionalscan only indicates one position at a time as ‘most active’ and does notexplore the synergistic relationship between positions.

[0142] The invention will now be described by reference to the followingexamples.

Example 1

[0143] In this Example the proteolytic enzyme of interest is Der P1,which is found in house dust mite faeces. The example illustrates thesynthesis of a number of FRET compounds in which the suitable bond is anunsubstituted amide bond, their use as a library for screening forpotential is substrates of Der P1, and subsequent identification andsynthesis of active inhibitors of the enzyme.

[0144] Purification of Der pI.

[0145] Crude mite extract (-100 mg, SmithKline-Beecham, U.K) wasdissolved in SmL Phosphate Buffered Saline (PBS; 50 mM potassiumphosphate; pH 7.4 containing 150 mM NaCl Der pI was purified by affinitycolumn chromatography using 4Cl antibody (indoor Biotechnology, Deeside,U.K.) The crude preparation was mixed with −2 mL of affinity resin for 2h at 4° C. and then washed with 2-3 volumes of PBS. Elution of boundprotein was carried out using 5 mM glycine containing 50% (v/v) ethyleneglycol. Fractions (2.2 mL) were collected and neutralised with 0.8 mL of0.2 M sodium phosphate buffer, pH 7.0. The fractions were pooled anddialysed overnight against 4 L PBS followed by a second dialysis against2 L PBS for 2-3 h. The total protein was concentrated as required byultrafiltration (MacroSep; Flowgen, U.K.)

[0146] Synthesis of Compounds

[0147] The compounds were synthesised using the Multipinapproach^(25,26) using Fmoc-Rink amide Macro crowns (Chiron MimotypesPty., Ltd., 11 Duerdin Street, Clayton Victoria 3168, Australia) with aloading of 7 μMoles.

[0148] The amino acid residues of each of the compounds were linkedusing amide bonds in a suitable form. The coupling chemistry employed issimilar to that reported in the literature²⁷ forfluorenylmethoxycarbonyl protected amino is acids and activatedpentafluorophenyl esters, in which the side-chains are protected usingacid labile protecting groups known to those skilled in the art, such asBoc- (for the —NH₂ of Lysine, —NH₂ of anthranilic acid and guanidino ofarginine), tBu- (for the —OH groups of serine, threonine and tyrosine),t-Bu for the —COOH group of Aspartic acid and Glutamic acid, Trityl-(for the Amide of Asparagine and Glutamine, and the amine functionalityof the Histidine ring.

[0149] The N-α-fluorenylmethoxycarbonyl protecting group of the coupledresidues were cleaved using 20% piperidine in dimethylformamide (DMF)for 30 minutes at 20° C. The coupling reactions for the free acids suchas Boc-ABz-OH (Boc-2-aminobenzoic acid), and Fmoc-(3-nitro)tyrosine-OHwere accomplished using 10 equivalents of a mixture of the free acid (1eq.):TBTU (0.98 eq.): HOBt (0.98 eq.): N-methylmorpholine (1.96 eq.) indimethylformamide (500 μL) as solvent for 5 hours at 20° C. The otheramino acids were coupled as their pentafluorophenyl esters²⁶ for 2-6hours.

[0150] Hence, in order to couple approximately equal ratios of eachcomponent in the mixture of the derivatised amino acids as theirpentafluorophenyl esters, a solution of a total of 0.98 equivalents(relative to the amino group loading on the crown) of the mixture ofamino acid pentafluorophenyl esters:HOBT (1 eq.) in DMF (500 μL) werecoupled for 16 hours at 20° C. The pins were then washed well with DMFand then recoupled using the same mixture under the same conditions. Athird coupling of 10 equivalents (relative to the amino group loading ofthe crown) for 2 hours in DMF was performed using this coupling protocolwith equimolar mixtures of the derivatised pentafluorophenyl esters ofthe amino acids in slightly less than 1 equivalent, it is possible toobtain approximately equal amounts of the coupled products to the crown.In this fashion the libraries are constructed with compounds present oneach crown. The compounds were cleaved from the crowns directly into the80 designated wells of the desired 96 well plate. In the cleavageprotocol each crown was treated with a mixture (600 μL) containingtrifluoroacetic acid (95%), triethylsilane (5%) for 2 hours at 20° C.The crowns were then washed with trifluoroacetic acid (500 μL) and thiswas then combined with the cleavage solution.

[0151] The Fmoc-Rink amide Macro crowns (Chiron Mimotypes Pty., Ltd., 11Duerdin Street, Clayton Victoria 3168, Australia) at 7 μMol loading percrown, were coupled with a 10 fold excess of a mixture containingL-Fmoc-Asp(O-t-Bu)-OH (leg) using TBTU (0.98 eq) and N-methylmorpholine(1.96 eq.) in the presence of HOBt (0.98 eq.) in DMF at 0.14Mconcentration. After deblocking of the Fmoc group with 20% piperidine inDMF for 30 minutes and subsequent washing with DMF and then methanol,coupling of the Fmoc-(3-nitro)tyrosine-OH was accomplished using 10equivalents of a mixture of the Fmoc-(3-nitro)tyrosine-OH (1 eq.):TBTU(0.98 eq.): HOBt (0.98 eq.): N-methylmorpholine (1.96 eq.) indimethylformamide as solvent at 0.14 M concentration for 5 hours at 20°C. Removal of the Fmoc group (vide infra) was followed by coupling ofthe mixtures of amino acids in the ratios outlined and under theconditions described (vide infra).

[0152] In a particular example the amino acids comprising group Binclude Ala, Val, Leu, Ser, Asn, Gln, Glu, Lys, Phe, Pro. The aminoacids comprising group C include Ala, Val, Leu, Ser, Asn, Gln, Glu, Lys,Phe, Pro. The amino acids comprising group D include Ala, Val, Ile, Leu,Nle, Ser, Glu, Phe. For n=4, the amino acids comprising group E includeAla, Val, Ile, Leu, Nle, Ser, Glu, Phe. Otherwise any selection from theamino acids can be made for n=1, 2, or 3.

[0153] The plates containing the combined cleavage solutions and werethen evaporated to dryness to yield the component mixtures using arotary centrifuge (“SPEEDVAC”, Savant Instruments Inc., Farmingdale,N.Y.) at 800 rpm for 1 hour at 20° C. under a reduced pressure of 10⁻²mmHg. Each component was then transferred to the final mother plateusing a (50%; 45%: 5%) mixture of acetonitrile:water:acetic acid. Theplates were then lyophilised to dryness using at 20° C. under a reducedpressure of 10⁻² mmHg, and then stored at −20° C. In this fashionlibraries of the type shown in FIGS. 2-14 were prepared.

[0154] In further detail, the Multipin approach which was employed isdescribed below:

[0155] Multipin Synthesis of Potential Substrates of Der pI

[0156] The ‘Chiron’ multipin kit consists of a standard 8×12 pin holdercontaining 96 ‘pin stems’ to which are reversibly attached ‘crowns’. The‘crowns’ provide a reactive polymer surface upon which a growing peptideis anchored during solid phase peptide synthesis. Each crown (theequivalent of the peptide-resin in standard solid phase synthesis) canbe considered to be an independent reactor by performing simultaneoussynthesis in individual 1 mL wells of industry standard 96 well plates.Each well, and thus each crown, can be charged with a unique set ofreagents providing unique sequences to each crown. Common steps such aswashing or removal of Nα protection can be performed concomitantly.

[0157] Synthesis is based upon the use of Nα-fluorenylmethyloxycarbonyl(Fmoc) protected amino acids. Side-chains of tri-functional amino acidsare protected with acid labile groups such as trityl or tert-butyl. Theaddition of amino-acid residues to the growing peptide chain, a processtermed ‘coupling’ proceeds through the utilisation of pre-formedpentafluorophenyl (pfp) esters or activation of the free acid, using thereagents HBTU or BOP in the presence of tertiary base (NMM) and HOBt ascatalyst.

[0158] The experimental techniques used are fully documented (Maeji,N.J. Bray, A. M. Valerio, R. M. and Wang, W., Peptide Research, 8(1),33-38, 1995 and Valerio, R. M. Bray, A. M. and Maeji, N. J. Int. J.Pept. Prot. Res, 44, 158-165, 1994) and the main steps are briefly asfollows.

[0159] General Methods

[0160] Preparation of Multipin Assembly

[0161] Whilst wearing standard plastic gloves, Fmoc-Rink Amidederivitized macrocrowns are assembled (simply clipped) onto stems andslotted into the 8×12 stem holder in the desired pattern for synthesis.

[0162] Removal of Nα-Fmoc Protection

[0163] A 250 mL solvent resistant bath is charged with 200 ml of a 20%piperidine/DMF solution. The multipin assembly is added and deprotectionallowed to proceed for 30 minutes. The assembly is then removed andexcess solvent removed by brief shaking. The assembly is then washedconsecutively with (200 mL each), DMF (5 mins) and MeOH (5 mins, 2 mins,2 mins) and left to air dry for 15 mins.

[0164] Quantitative UV Measurement of Fmoc Chromophore Release

[0165] A 1 cm path length UV cell is charged with 1.2 mL of a 20%piperidine/DMF solution and used to zero the absorbance of the UVspectrometer at a wavelength of 290 nm. A UV standard is then preparedconsisting of 5.0 mg Fmoc-Asp(OBut)-Pepsyn KA (0.08 mmol/g) in 3.2 mL ofa 20% piperidine/DMF solution. This standard gives Abs₂₉₀=0.55-0.65 (atRT). An aliquot of the multipin deprotection solution is then diluted asappropriate to give a theoretical Abs₂₉₀=0.6, and this value comparedwith the actual experimentally measured absorbance showing theefficiency of previous coupling reaction.

[0166] Coupling of Amino-Acid Residues

[0167] Whilst the multipin assembly is drying, the appropriate Nα-Fmocamino acid pfp esters (10 equivalents calculated from the loading ofeach crown) and HOBt (10 equivalents) required for the particular roundof coupling are accurately weighed into suitable containers.Alternatively, the appropriate Nα-Fmoc amino acids (10 equivalentscalculated from the loading of each crown), desired coupling agent e.g.HBTU (9.9 equivalents calculated from the loading of each crown) andactivation eg HOBt (9.9 equivalents calculated from the loading of eachcrown), NMM (19.9 equivalents calculated from the loading of each crown)are accurately weighed into suitable containers.

[0168] The protected and activated Fmoc amino acid derivatives are thendissolved in DMF (500 μl for each macrocrown, e.g. for 20 macrocrowns,20×10 eq×7 mmoles of derivative would be dissolved in 10 000 μL DMF).The appropriate derivatives are then dispensed to the appropriate wellsready for commencement of the ‘coupling cycle’. As a standard, couplingreactions are allowed to proceed for 2-6 hours (depending upon nature ofcoupling e.g. Ala to Ala 2 hours Val to Leu 6 hours.

[0169] When coupling Fmoc amino-acid pentafluorophenyl esters, 10 eq ofderivative in DMF (400 μl) with bromophenol blue stock solution (100 μl)is used for each macrocrown. This allows monitoring of the progress ofthe acylation reaction through the disappearance of the deep bluecoloration of bromophenol blue in the presence of unreacted amine to apale yellow upon completion of acylation.

[0170] Preparation of Bromophenol Blue Stock Solution

[0171] Bromophenol blue (20 mg) is dissolved in DMF (50 mL) and HOBt (10mg) added.

[0172] Washing Following Coupling

[0173] If a 20% piperidine/DMF deprotection is to immediately follow thecoupling cycle, then the multipin assembly is briefly shaken to removeexcess solvent washed consecutively with (200 mL each), MeOH (5 mins)and DMF (50 mins) and deprotected (see above). If the multipin assemblyis to be stored, then a full washing cycle consisting brief shaking thenconsecutive washes with (200 mL each), DMF (5 mins) and MeOH (5 mins, 2mins, 2 mins) is performed.

[0174] Acidolytic Mediated Cleavage of Peptide-Pin Assembly

[0175] Acid mediated cleavage protocols are strictly performed in a fumehood. A polystyrene 96 well plate (1 mL/well) is labelled, then the tareweight measured to the nearest mg. Appropriate wells are then chargedwith a trifluoroacetic acid/triethylsilane (95:5, v/v, 600 μl) cleavagesolution, in a pattern corresponding to that of the multipin assembly tobe cleaved.

[0176] The multipin assembly is added, the entire construct covered intin foil and left for 2 hrs. The multipin assembly in then added toanother polystyrene 96 well plate (1 mL/well) containing trifluoroaceticacid/triethylsilane (95:5, v/v, 600 μl) (as above) for 5 mins.

[0177] The cleaved assembly is washed with DMF (200 μL, 5 mins), MeOH(200 μL, 5 mins), the spent crowns removed and discarded, the stemsremoved and washed by sonication in methanol (1 hr, RT).

[0178] Work Up of Cleaved Peptides

[0179] The primary polystyrene cleavage plate (2 hr cleavage) and thesecondary polystyrene plate (5 min wash) (see above) are then placed inthe SpeedVac and the solvents removed (minimum drying rate) for 90 mins.

[0180] The contents of the secondary polystyrene plate (see above) aretransferred to their corresponding wells on the primary plate using anacetonitrile/water/acetic acid (50:45:5, v/v/v) solution (3×150 μl) andthe spent secondary plate discarded.

[0181] Analysis of Products

[0182] 1.0 μl of each well (see above) is diluted to 400 μl with 0.1% aqTFA and analysed by HPLC-MS. Column Vydac C4 (214TP52, narrowbore,21×250 mm). Eluents:—Solvent A 0.1% aq trifluoroacetic acid, SolventB=acetonitrile/10% A. Gradient:—10-90% B in A over 27 mins, 250 ml/min,215 nm UV detection. The individual substrates described below wereprepared by the above methods and shown by HPLC-MS to be>95% with thecorrect mass.

[0183] Final Lyophilisation of Peptides

[0184] The primary polystyrene plate (plus the washings from thesecondary plate) is covered with tin foil, held to the is plate with anelastic band. A pin prick is placed in the foil directly above each welland the plate placed at −80° C. for 30 mins. The plate is thenlyophilised on the ‘Heto freeze drier’ overnight. Where appropriateindividual peptides were then weighed and dissolved to 10 mM stocksolutions in DMSO prior to biological screening. Alternatively the 20component mixture is weighed and the peptide/20 component ratio iscalculated.

[0185] Further coupling of amino acid residues was carried out accordingto the multipin approach described above. Whilst the multipin assemblywas drying, the appropriate Nα-Fmoc amino acids (10 equivalentscalculated from the loading of each crown), HATU coupling agent (9.9equivalents calculated from the loading of each crown), HOAt catalyst(9.9 equivalents calculated from the loading of each crown) and DIPEA(19.9 equivalents calculated from the loading of each crown)wereaccurately weighed into suitable containers.

[0186] The protected Nα-Fmoc amino acids and coupling agents were thendissolved in DMF (500 μl for each macrocrown) and activated by theaddition of DIPEA. The appropriate derivatives were then dipensed totheir appropriate wells and as standard coupling to each macro crown wasallowed to proceed for 2 hours.

[0187] When coupling particularly hindered amino acid residues such asN-Methyl, Ca-Methyl or unusual amino acids (whose coupling efficiency isunknown) the coupling reaction was repeated, as standard, for a further2 hours.

[0188] Substrates for Der pI

[0189] Using the general techniques described above, the followingcompounds were prepared and assayed as potential substrates against DerpI purified as described above. Meas- ured K_(m) Peptide [SEQ ID Nos.1-76] (μM) Abz-Val-Ala-Nle-Ser-Tyr (NO₂) -Asp-NH₂ 12H-Val-Ala-Nle-Ser-TyrNO₂-ASp-NH₂ NS H-Ala-Nle-Ser-Tyr (NO₂)-Asp-NR2 NSAc-Val-Ala-Nie-Ser-Tyr (NO₂)-Asp-NH₂ NS H-Val-Ala-Nle-Ser-Tyr (NO₂)-NH₂NS H-Ala-Nle-Ser-Tyr (NO₂)-ASP-NH₂ NS Ac-Val-Ala-Nle-Ser-Tyr (NO₂)-NH₂NS Abz-Val-Ala-Nle-Ser-Tyr (NO₂)-NH₂ NM Abz-Val -Ala-Nle-Ser-NH₂ NMAbz-Val-Ala-Nle-Ser-Phe-Asp-NH₂ NM Abz-Val-Ala-Nle-Ser-Tyr-Asp-NH₂ NMAbz-Val-Ala-Nle-Ser-Ala-Asp-NH₂ NM Abz-Val-Ala-Nle-Ser-Lys-Asp-NH₂ NMAbz-Val-Ala-Nle-Ser-eAHA-Asp-NH₂ NM Abz-Ala-Nle-Ser-Tyr(NO₂)-Asp-NH₂ NMAbz-Nle-Ser-Tyr(NO₂)-Asp-NH₂ NS Bz-Val-Ala-Nle-Ser-Tyr (NO₂)-NH₂  NM^(a)Bz(2-carboxy)-Val-Ala-Nle-Ser-Tyr(NO₂)-NH₂  NM^(a)Chex-Val-Ala-Nle-Ser-Tyr (NO₂)-NH₂  NM^(a) n-Bu-Val-Ala-Nle-Ser-Tyr(NO₂)-NH₂  NM^(a) Piv-Val-Ala-Nle-Ser-Tyr (NO₂)-NH₂  NM^(a)Bz-Val-Ala-Nle-Ser-Tyr (NO₂)-NH₂  NM^(a) Abz-Val-Ala-Lys-Ser-Tyr(NO₂)-Asp-NH₂ 14 Abz-Val-Ala-Gln-Ser-Tyr(NO₂)-Asp-NH₂ 6Abz-Val-Ala-Thr-Ser-Tyr (NO₂)-Asp-NH₂ 6 Abz-Val-Ala-hLeu-Ser-Tyr(NO₂)-Asp-NH₂ 4 Abz-Val-Ala-Cha-Ser-Tyr (NO₂)-Asp-NH₂ 5Abz-Val-Ala-His-Ser-Tyr (NO₂)-Aso-NH₂ >20 Abz-Val-Ala-ACH-Ser-Tyr(NO₂)-Asp-NH₂ NS Abz-Val-Ala-DNle-Ser-Tyr (NO₂)-Asp-NH₂ NSAbz-Val-Ala-3pyr-Ser-Tyr (NO₂)-Asp-NH₂ 10 Abz-Val-Ala-Hyp-Ser-Tyr(NO₂)-Asp-NH₂ NS Abz-Val-Ala-ACP-Ser-Tyr (NO₂)-Asp-NH₂ NSAbz-Val-Lys-hLeu-Ser-Tyr (NO₂)-Asp-NH₂ 35 Abz-Val-+E,unlDAla-hLeu-Ser-Tyr (NO₂)-Asp-NH₂ NS Abz-Val-Tic-hLeu-Ser-Tyr(NO₂)-Asp-NH₂ NS Abz-Val-ACH-hLeu-Ser-Tyr (NO₂)-Asp-NH₂ NSAbz-Val-Met(O)-hLeu-Ser-Tyr (NO₂)-Asp-NH₂ 35 Abz-Val-2Nal-hLeu-Ser-Tyr(NO₂)-Asp-NH₂ NS Abz-Val-ACP-hLeu-Ser-Tyr (NO₂)-Asp-NH₂ NSAbz-Val-DLys-hLeu-Ser-Tyr (NO₂)-Asp-NH₂ NS Abz-Val-DGln-hLeu-Ser-Tyr(NO₂)-Asp-NH₂ NS Abz-Val-3pyr-hLeu-Ser-Tyr (NO₂)-Asp-NH₂ NSAbz-Val-Cha-hLeu-Ser-Tyr (NO₂)-Asp-NH₂ NS Abz-DVal-Ala-hLeu-Ser-Tyr(NO₂)-Asp-NH₂ NS Abz-Gln-Ala-hLeu-Ser-Tyr (NO₂)-Asp-NH₂ 12Abz-Lys-Ala-hLeu-Ser-Tyr (NO₂)-Asp-NH₂ >15 Abz-Tic-Ala-hLeu-Ser-Tyr(NO₂)-Asp-NH₂ NS Abz-ACH-Ala-hLeu-Ser-Tyr (NO₂)-Asp-NH₂ NSAbz-Met(O)-Ala-hLeu-Ser-Tyr(NO₂)-Asp-NH₂ 20 Abz-3pyr-Ala-hLeu-Ser-Tyr(NO₂)-Asp-NH₂ >10 Abz-2Nal-Ala-hLeu-Ser-Tyr (NO₂)-Asp-NH₂ 15Abz-Leu-Ala-hLeu-Ser-Tyr (NO₂)-Asp-NH₂ 18 Abz-Cha-Ala-hLeu-Ser-Tyr(NO₂)-Asp-NH₂ 9 Abz-Bip-Ala-hLeu-Ser-Tyr (NO₂)-Asp-NH₂ 2.5Abz-Bip-Ala-hLeu-Tyr-Tyr (NO₂)-Asp-NH₂ 3 Abz-Bip-Ala-hLeu-Leu-Tyr(NO₂)-Asp-NH₂ 3.7 Abz-Bip-Ala-hLeu-Lys-Tyr (NO₂)-Asp-NH₂ 2Abz-Bip-Ala-hLeu-Asp-Tyr (NO₂)-Asp-NH₂ 5.0 Abz-Bip-Ala-hLeu-Abu-Tyr(NO₂)-Asp-NH₂ 1.7 Abz-Bip-Ala-hLeu-Cha-Tyr (NO₂)-Asp-NH₂ 2.5Abz-Bip-Ala-hLeu-Met(O)-Tyr (NO₂)-Asp-NH₂ 5 Abz-Bip-Ala-hLeu-Thr-Tyr(NO₂)-Asp-NH₂ 2.5 Abz-Bip-Ala-hLeu-3pyr-Tyr (NO₂)-Asp-NH₂ 4Abz-Bip-Ala-hLeu-Bu^(t)Gly-Tyr (NO₂)-Asp-NH₂ 4 Abz-Bip-Ala-hLeu-Hyp-Tyr(NO₂)-Asp-NH₂ 4 Abz-Phe-Val-Ala-Nle-Ser-Tyr (NO₂)-Asp-NH₂ NMAbz-3.Pyr-Val-Ala-Nle-Ser-Tyr (NO₂)-Asp-NH₂ NMAbz-1.Naph-Val-Ala-Nle-Ser-Tyr (NO₂)-Asp-NH₂ 17Abz-2.Naph-Val-Ala-Nle-Ser-Tyr (NO₂)-Asp-NH₂ NMAbz-Tyr-Val-Ala-Nle-Ser-Tyr(NO₂)-Asp-NH₂ Abz-Bip-Val -Ala-Nle-Ser-Tyr(NO₂)-Asp-NH₂ 10 Abz-Lys-Val-Ala-Nle-Ser-Tyr(NO₂)-Asp-NH₂ 15Abz-Glu-Val-Ala-Nle-Ser-Tyr (NO₂)-Asp-NH₂ 20 Abz-Leu-Val-Ala-Nle-Ser-Tyr(NO₂)-Asp-NH₂ Abz-Hyp-Val-Ala-Nle-Ser-Tyr (NO₂)-Asp-NH₂ NS

[0190] Ranked in order of cleavage rate:-Bz>n-But>Piv>Bz(2-carboxy)>Abz.

[0191] Coupling of amino acid residues was carried out according to themultipin approach described above. Whilst the multipin assembly wasdrying, the appropriate Nα-Fmoc amino acids (10 equivalents calculatedfrom the loading of each crown), HATU coupling agent (9.9 equivalentscalculated from the loading of each crown), HOAt catalyst (9.9equivalents calculated from the loading of each crown) and DIPEA (19.9equivalents calculated from the loading of each crown) were accuratelyweighed into suitable containers.

[0192] The protected Nα-Fmoc amino acids and coupling agents were thendissolved in DMF (500 μl for each macrocrown) and activated by theaddition of DIPEA. The appropriate derivatives were then dispensed totheir appropriate wells and standard coupling to each macrocrown wasallowed to proceed for 2 hours.

[0193] When coupling particularly hindered amino acid residues such asN-Methyl, Ca-Methyl or unusual amino acids (whose coupling efficiency isunknown) the coupling reaction was repeated, as standard, for a further2 hours.

[0194] The following sequences were synthesised in this way: MeasuredPeptide Sequence [SEQ ID Nos. 77-85] Km(μM)Abz-Val-Ala-(NMe)Nle-Ser-Tyr(NO₂)-Asp-NH₂ NSAbz-Val-(NMe)Ala-Nle-Ser-Tyr(NO₂)-ASP-NH₂ NSAbz-Val-Ala-Aib-Ser-Tyr(NO₂)-Asp-NH₂ NSAbz-Val-Aib-Nle-Ser-Tyr(NO₂)-Asp-NH₂ NSAbz-Deg-Ala-Nle-Ser-Tyr(NO₂)-Asp-NH₂ NSnBu-D.Ser-D.Nle-D.Ala-D.Val-p.Aba-NH₂ NS Bz-Val-Ala-Statine-Ser-eAha-NH₂NS Abz-p.Aba-Nle-Ser-Tyr(NO₂)-Asp-NH₂ NSAbz-Cmpi-Nle-Ser-Tyr(NO₂)-ASP-NH₂ NS

[0195] Assay Procedure

[0196] Each mixture of 20 compounds in the libraries of the apparatusdescribed herein was screened at a concentration of 1.0 μM per compoundin an assay against the cysteinyl protease Der pI. The most active wellswere identified by the rate of emission of fluorescence at 420 nm whenthe samples were irradiated at 320 nm. An analysis of the twocomplementary libraries showed that the best substrates for the enzymewere:

[0197] Abz-B-C-D-E-Tyr(NO2)-Asp-NH2

[0198] Where

[0199] B=Valine>Alanine, Glutamine, Leucine, Phenylalanine

[0200] C=Alanine>>Glutamine, or Lysine.

[0201] D=Leucine, Norleucine or Alanine>Serine

[0202] E=Serine

[0203] The best substrate was:

[0204] [SEQ ID:1] Abz-Val-Ala-Nle-Ser-Tyr(NO₂) -Asp-NH₂

[0205] This compound was then resynthesised as a single component usingthe peptide synthesis methodology described herein. The k_(cat)/K_(m)value for the pure substrate in the Der pI assay was measured as3.5×10⁴M⁻¹s⁻¹, and was considered to be suitably high for use in a highthroughput assay for the general screening of inhibitors of Der pI.

[0206] High Throughput Assay Development

[0207] Plate assays were carried out in 96 well plate format, using 0.1ug of Der pI per 100 μL assay volume in each well and using 20 μM of thesubstrate. All assays were performed in Assay Buffer (AB; 50 mMpotassium phosphate, pH 8.25 containing 1 mM ethylenediaminetetraaceticacid (EDTA) and 1 mM dithiothreitol (DTT). The Der pI enzyme ispre-activated by addition of DTT and this in incubated at roomtemperature for 5 min. prior to initiation of the assay. As an examplefor the screening methodology, each well contains a 5 μL of a 20 μMsolution of the test compound in DMSO, 10 μL of a 200 μM aqueoussolution of the substrate, 2, and 85 μL of Der pI in AB is added toinitiate the reaction. Enzyme activity is monitored by fluorescenceusing 320 nm for excitation and 420 nm for the emission wavelengthsusing a Labsystems Fluroskan Ascent machine. Kinetic measurements werecarried out using a Hitachi F-4500 Fluorescence Spectrophotometer.

[0208] Synthesis of Inhibitors of Der pI

[0209] The best substrate described above was shown by HPLC-massspectroscopic analysis of the enzyme/assay solution, to be cleavedbetween the Norleucine-Serine amide bond. Replacement of the terminalAbz group by a series of derivatives (e.g. Poc-, Pivaloyl, Benzoyl, and2-carboxy-Benzoyl) affected substrate activity and specificity for theDer pI enzyme. With this knowledge of the P₁-P₁′ cleavage site and forthe P₄-P₃-P₂-P₁ motif, the compound Boc-Val-Ala-Leu-H, 4, wassynthesised as shown in Scheme 1a, FIG. 15.

[0210] Attachment of a suitable Michael acceptor such as CH═CH—CO2Et,and —CH═CH—SO₂ Ph to the motif (Scheme 2, FIG. 16), provided activeinhibitors of the enzyme with apparent IC₅₀ values of 50 nM, 1000 nM and100 nM respectively.

[0211] Preparation of an Acyloxymethylketone Series

[0212] A series of acyloxymethylketone compounds having active Der PIinhibitor activity was prepared by the following procedures:

[0213] N-Benzoyl-L-valyl-L-alanyl-L-norleucine

[0214] N-Benzoyl-L-valyl-L-alanyl-L-norleucine was prepared by solidphase benzoylated peptide synthesis as follows:

[0215] Resin Loading (Step 1)

[0216] 2-Chlorotritylchloride resin (4.9 g, 1.05 mmol/g, Novabiochem)was swelled in dichloromethane (40 ml) and a suspension ofFmoc-L-norleucine added and stirred for 5 minutes. A solution ofdiisopropylethylamine in DCM(10 ml, 57 mmol in 30 ml) was added over 5minutes and the resulting mixture stirred at room temperature for 2hours. Methanol (5 ml) added and reaction mixture stirred for a further10 minutes before resin filtered and washed with 3×DCM, 2×DMF,2×2-propanol, 2×DMF, 2×2-propanol, methanol, 2×ether and dried undervacuum for 24 hours.

[0217] Amino Acid Deprotection (Step 2)

[0218] Fmoc-L-norleucine loaded resin was deprotected by 20 treatmentwith 20% piperidine in DMF over 4 hours. The swollen resin was filtered,washed with 5×DMF, 2×ether and dried under vacuum for 24 hours.

[0219] Peptide Chain Extension (Step 3)

[0220] L-Norleucine loaded resin (5 mmol) was added to a solution ofFmoc-L-alanine (6.23 g, 20 mmol), hydroxybenzotriazole (3.0 g, 20 mmol),2-(1-H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (7.59 g, 20 mmol) and diisopropylethylamine (6.97ml, 40 mmol) in DMF (20 ml) and allowed to swell over 4 hours with mildagitation. Resin was filtered and washed with 4×DMF, 2×ether and driedunder vacuum overnight.

[0221] Steps (2) and (3) were carried out repetitively withFmoc-L-alanine and Fmoc-L-valine to afford resin bound tripeptideH-L-valyl-L-alanyl-L-norleucine.

[0222] Peptide Chain Benzoylation (Step 4)

[0223] L-Valyl-L-alanyl-L-norleucine loaded resin (1 g, approx. 1 mmol)was added to a solution of benzoic acid (0.488 g, 4 mmol),hydroxybenzotriazole (0.6 g, 4 mmol),2-(1-H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate (1.52 g, 4 mmol) and diisopropylethylamine (1.40 ml,8 mmol) in DMF (5 ml) and allowed to swell over 6 hours with mildagitation. Resin was filtered and washed with 4×DMF, 2×ether and driedunder vacuum overnight.

[0224] Resin Cleavage (Step 5)

[0225] N-Benzoyl-L-valyl-L-alanyl-L-norleucine loaded resin (1.0 g,approx. 1 mmol) was treated with a 1% solution of trifluoroacetic acidin dichloromethane (20 ml) containing triethylsilane (320 ml, 2 mmol)for 1 hour. Resin was removed by filtration and washed withdichloromethane (3×10 ml). Organic layer was collected, evaporated andtitrated with ether to affordN-benzoyl-L-valyl-L-alanyl-L-norleucine.(285 mg). Electrospray-MS m/z407 [MH⁺].

[0226] Bromomethylketone Formation (Step 6)

[0227] N-Benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketoneN-Benzoyl-L-valyl-L-alanyl-L-norleucine(140 mg, 0.34 mmol) was suspendedin dry THF(3 ml) and dry DMF was added dropwise to afford homogeneity.The reaction mixture was cooled to-10° C. and isobutylchloroformate (129ml, 11.0 mmol) and N-methylmorpholine (109 ml, 1.0 mmol) added withstirring under Argon. The mixture was stirred for 30 minutes before asolution of diazomethane in ether(5 ml, approx. 2 mmol) was added. Thereaction mixture was allowed to warm to room temperature over 1 hourbefore a 1:1 solution of is acetic acid and 50% HBr (1 ml, 3.0 mmol HBr)was added dropwise and stirred for 15 minutes. The organic phase wasdiluted with ethylacetate (40 ml), washed with water (10 ml), brine (10ml) and sodium bicarbonate (2×10 ml), dried over MgSO⁴ solvent removedunder vacuum. This afforded an off white solid (152 mg) which could befurther purified as required by prep. HPLC. Electrospray-MS m/z 482[MH⁺] and 484 [MH⁺].

[0228] Acyloxymethylketone Formation (Step 7)

[0229] N-Benzoyl-L-valyl-L-alanyl-L-norleucine 2,6-bis(trifluoro methyl)Benzoyloxymethyl Ketone

[0230] A mixture of potassium fluoride (0.1 mmol, 6 mg) and2,6-bis(trifluoromethyl)benzoic acid (0.066 mmol, 17 mg) in dry DMF (500ml) was stirred over molecular sieves at room temperature for 5 minutes.A solution of N-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone(0.033 mmol, 16 mg) in dry DMF (500 ml) was added and the reactionmixture stirred for 1 hour. The reaction mixture was passed through ashort silica plug and washed with 5% methanol in dichloromethane.Solvent was removed under vacuum and the residue purified using prep.HPLC. Freeze drying afforded (6.4 mg) as a white lyophilisate.Electrospray-MS m/z 660 [MH⁺].

[0231] 10 Similarly the following compounds were prepared:

[0232] N-Benzoyl-L-valyl-L-alanyl-L-norleucine2,6-dimethylbenzoyloxymethyl ketone (Electrospray-MS m/z 552 [MH⁺]) fromof N-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and2,6-dimethylbenzoic acid.

[0233] N-Benzoyl-L-valyl-L-alanyl-L-norleucine 2-hydroxybenzoyloxymethylketone (Electrospray-MS m/z 540 [MH⁺] from ofN-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and2-hydroxybenzoic acid.

[0234] N-Benzoyl-L-valyl-L-alanyl-L-norleucine2,6-dichlorobenzoyloxymethyl ketone (Electrospray-MS m/z 592 [MH⁺] and594 [MH⁺] from of N-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethylketone and 2,6-dichlorobenzoic acid.

[0235] N-Benzoyl-L-valyl-L-alanyl-L-norleucine benzoyloxymethyl ketone(Electrospray-MS m/z 524 [MH⁺]) from ofN-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and benzoicacid.

[0236] N-Benzoyl-L-valyl-L-alanyl-L-norleucine2,3,4,5,6-pentafluorobenzoyloxymethyl ketone (Electrospray-Ms m/z 614[MH⁺]) from of N-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethylketone and 2,3,4,5,6-pentafluorobenzoic acid.

[0237] N-Benzoyl-L-valyl-L-alanyl-L-norleucine1,1-dimethylpropyloxymethyl ketone (Electrospray-MS m/z 504 [MH⁺])fromof N-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and1,1-dimethylpropanoicacid.

[0238] N-Benzoyl-L-valyl-L-alanyl-L-norleucineN(-benzyloxycarbonyl)-D-serinyl-(O-tert-butyl)oxymethyl ketone(Electrospray-MS m/z 697 [MH⁺])from ofN-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone andN-benzyloxycarbonyl-D-serine-O-tert-butylether.

[0239] N-Benzoyl-L-valyl-L-alanyl-L-norleucineN(-benzyloxycarbonyl)-D-serineoxy methyl ketone (Electrospray-MS m/z 641[MH⁺])from of N-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketoneand N-benzyloxycarbonyl-D-serine.

[0240] N-Benzoyl-L-valyl-L-alanyl-L-norleucine 2-furanoxy methyl ketone(Electrospray-MS m/z 514 [MH⁺])from ofN-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and 2-furancarboxylic acid.

[0241] N-Benzoyl-L-valyl-L-alanyl-L-norleucine 2,6-dichlorophenylacyloxymethyl ketone (Electrospray-MS m/z 606 [MH⁺], 608 [MH⁺]) from ofN-benzoyl-L-valyl-L-alanyl-L-norleucine bromomethyl ketone and2,6-dichlorophenylacetic acid.

[0242] Standard Prep. HPLC conditions were as follows: C4 preparativeHPLC system (Vydac, 22×250 mm) eluting at 10 ml per minute a gradient of5-95% (90% acetonitrile (0.1% TFA)) over 30 minutes.

[0243] The following compounds were prepared by the techniques andprocedures described beneath each named compound.

[0244] Preparation of Ethyl-(S)-(E)-3-((tert-butoxy Carbonyl Amino ValylAlanyl) Amino-6-methyl-hept-2-enoate

[0245] To a suspension of sodium hydride (46 mg, 1.9 mmol) in anhydrousTHF (4 ml) cooled to 0° C. was added a solution oftriethylphosphonoacetate (420 mg, 1.9 mmol) in THF (2 ml) dropwise over5 minutes and the mixture stirred until gas evolution ceased. Thesolution was added dropwise to a solution of BocVAL-CHO (600 mg, 1,56mmol) in dry THF cooled to −10° C. The reaction mixture was stirred for1 hour and saturated ammonium chloride (10 ml) was added. A white solidprecipitated which was removed by filtration and the filtrate waspartitioned between ethyl acetate and water. The organic layer was driedwith magnesium sulphate and evaporated to give an oil which wascrystallised from acetonitrile water to yield the title compound, 640mg, 91%.

[0246] MS (EI+ve) required (M⁺(C₂₃H₄₁N₃O₆)+1)=456: found(M⁺+H)=456,((M⁻-^(t)BOC)+1)=356 (100%).

[0247] Preparation of (S)-(E)-3-((tert-butoxy Carbonyl Amino ValylAlanyl) Amino-6-methyl-hept-2-enoic Acid

[0248] The ethyl ester (455 mg, 1 mmol) was dissolved in dioxane (10 ml)and water added followed by lithium hydroxide (126 mg, 3 mmol). Thesolution was stirred for 3 hours and 1M HCl aq was added until the pHreached neutrality. The dioxane was removed by rotary evaporation andthe pH adjusted to 4 with 1M HCl aq. The title compound precipitated,filtered and washed with water to yield 420 mg, 98%.

[0249] MS (EI+ve) required (M⁺(C₂₁H₃₇N₃O₆)+1)=428: found(M⁺+H)=428(100%).

[0250] Preparation of 1,1,1-Trifluoroethyl-(S)-(E)-3-((tert-ButoxyCarbonyl Amino Valyl Alanyl) Amino-6-methyl-hept-2-enoate

[0251] The acid (BocVAL-CO₂H) (50 mg, 0.117 mmol) anddimethylaminopyridine (29 mg, 0.24 mmol) was dissolved in drydichloromethane (1 ml) and cooled to 0° C. Water soluble carbodiimidehydrochloride salt (26 mg, 0.13 mmol) in 0.5 ml dichloromethane wasadded and the solution stirred for 5 minutes. 1,1,1-Trifluoroethanol(0.017 ml, 0.23 mmol) in 0.5 ml dichloromethane was added and thereaction was allowed to warm to room temperature after 1 hour and thereaction mixture stirred overnight. The reaction mixture was washed 2×2ml 0.5 M citric acid solution, 1×2 ml water, 1×2 ml saturated sodiumbicarbonate solution, 1×2 ml water, dried with magnesium sulphate andevaporated to dryness to give the title compound.

[0252] MS (EI+ve) required (M⁺(C₂₁H₃₈N₃O₆F₃)+1)=510: found(M⁺+H)=510,((M⁺-^(t)BOC)+1)=410, ((M⁺-^(t)Bu)+1)=454 (100%).

[0253] Preparation of Ethyl-(S)-(E)-3-(N-benzoyl Valyl Alanyl)Amino-6-methyl-hept-2-enoate

[0254] The tert-butoxy carbonyl protected ethyl ester (16.6 mg, 0.036mmol) was dissolved in 4.0M HCl in dioxane (2 ml) stirred at roomtemperature for 30 minutes and evaporated to dryness. The residue wasdissolved in DMF (0.5 ml) and N-methylmorpholine (7.36 mg, 0.073 mmol)added followed by benzoyl chloride (5.4 mg. 0.038 mmol) in DMF 0.5 ml.The reaction stirred for 2 hours, diluted with 0.1% trifluoroacetic acidsolution (4 ml) and acetonitrile (2 ml) and injected onto a C4preparative HPLC system (22×250 mm) eluting at 10 ml per minute,monitoring at 215 nm and a gradient of 10-90% system B over 25 minutesand holding at 90% for 15 minutes. System A=0.1% TFA in water, systemB=90% acetonitrile, 10% system A. The peak eluting at 26-28 minutes wascollected and lyophilised to a white solid, yield 4.5 mg, 27%.

[0255] Analysis by MS (EI+ve) required (M⁺(C₂₁H₃₇N₃O₅)+1)=460:found(M⁺+H)=460.

[0256] Preparation of Diethyl Phenylsulfonylmethylphosphonate

[0257] The Diethyl Phenyl sulfonylmethylphophonate was prepared using amethod adapted from I. Shahak, and J. Almog. (Synthesis, 145,1970).

[0258] The commercially available diethyl phenylthiomethylphosphonate(1.0 ml, 4.1 mmol) was dissolved in dichloromethane (10 ml). Sulphuricacid (10 ml, 25%) was added and the mixture cooled on ice. Solidpotassium permanganate was then added in three aliquots of 0.5 g withstirring. After the additions the reaction appeared to be complete.Solid sodium metabisulfite was added slowly until the mixture turnedcolourless. This was then extracted with ethyl acetate (×3) and thecombined organic washings washed with saturated sodium bicarbonatesolution followed by brine before drying over sodium sulphate. Thevolatiles were removed in vacuo. The residue was purified by flashchromatography on silica eluting initially with ethyl acetate/hexane 8/2followed by pure ethyl acetate.

[0259] In this way the desired product, diethylphenylsulfonylmethylphosphonate (1.0 9, quant) was obtained as acolourless solid.

[0260] The product was analysed by mass spectrometry (MS) (MALDI-TOF):required (M⁺(C₁₁H₁₇O₅PS)+1)=292; obtained (M⁺+1)=292

[0261] Preparation of(S)-(E)-3-((tert-butoxycarbonylamino-valyl)alanyl)amino-1-phenylsulfonyl-5-methyl-1-hexeneDiethyl phenylsulfonylmethylphosphonate (38 mg, 129 mmol) was dissolvedin dry THF (10 ml) and then cooled to 0° C. under an atmosphere ofnitrogen. Sodium hydride (8 mg of 60% dispersion in oil, 200 mmol) wasadded and the mixture stirred for 15 mins (effervescence). The aldehyde^(t)Boc-Val-Ala-Leu-CHO (50 mg, 129 mmol) was then added to theresulting solution and the mixture was stirred for 60 mins. The reactionwas quenched by the addition of dilute hydrochloric acid (0.1 M),followed by extraction with ethyl acetate (×3). The separated organicphase was sequentially washed with saturated sodium bicarbonate solutionand brine before drying over sodium sulphate. The volatiles were removedin vacuo. The residue was purified by flash chromatography on silicaeluting with ethyl acetate/hexane 4/6. An unidentified by-product waseluted first (12 mg) followed by the desired product(S)-(E)-3-((tert-butoxycarbonylamino-valyl)alanyl)amino-phenylsulfonyl-5-methyl-1-hexene(22 mg, 32%) as a solid.

[0262] MS (electrospray) required (M⁺(C₂₆H₄₁O₆N₃S)+1)=523: found(M⁺+Na)=546, ((M-^(t)Boc)+1)=424 (100%).

[0263] Preparation of Diethyl Methylsulfonylmethylphosphonate

[0264] The commercially available Diethyl methylthiomethylphosphonatewas converted to the title compound using the method of I. Shahak and J.Almog (Synthesis, 171, 1969).

[0265] Preparation of(S)-(E)-3-((tert-butoxycarbonylamino-valyl)alanyl)amino-1-methylsulfonyl-5-methyl-1-hexene

[0266] Diethyl methylsulfonylmethylphosphonate (30 mg, 130 mmol) wasdissolved in dry THF (5 ml) and then cooled to 0° C. under an atmosphereof nitrogen. Sodium hydride (7 mg of 60% dispersion in oil, 175 mmol)was added and the mixture stirred for 15 mins (effervescence). Thealdehyde ^(t)Boc-Val-Ala-Leu-CHO (50 mg, 129 mmol) was then added to theresulting solution and the mixture then stirred for 60 mins. Thereaction was quenched by addition of dilute hydrochloric acid (0.1 M),followed by extraction with ethyl acetate(×3). The separated organicphase was sequentially washed with saturated sodium bicarbonate solutionand brine before drying over sodium sulphate. The volatiles were thenremoved in vacuo. The residue was purified by flash chromatography onsilica eluting with ethyl acetate/hexane 8/2. An unidentified by-productwas eluted first (4 mg), followed by the desired product(S)-(E)-3-((tert-butoxycarbonylamino-valyl)alanyl)amino-methylsulfonyl-5-methyl-1-hexene(24 mg, 40%) as a solid.

[0267] MS (electrospray) required (M⁺(C₂₁H₃₉O₆N₃S)+1)=462: found(M⁺+Na)=484, ((M-^(t)Boc)+1)=362 (100%).

[0268] Preparation of Ethyl Diethylphosphorylmethylsulfonate

[0269] Prepared in accordance with procedure B described in L. Ghosezet. al. (Tetrahedron, 43, 5125, 1987).

[0270] The product was analysed on MS (electrospray) required(M⁺(C₇H₁₇O₆PS)+1)=261: Found (M⁺+H)=261, (M⁺+Na)=283.

[0271] Preparation ofDiethyl(S)-(E)-3-((tert-butoxycarbonylamino-valyl)alanyl)amino-5-methylhexenylsulfonate.

[0272] Ethyl diethylphosphorylmethanesulfonate (36 ml, −138 mmol) wasdissolved in dry THF (5 ml) and then cooled to 0° C. under an atmosphereof nitrogen. Sodium hydride (8 mg of 60% dispersion in oil, 200 mmol)was added and the mixture stirred for 15 mins (effervescence). Thealdehyde ^(t)Boc-Val-Ala-Leu-CHO (50 mg, 129 mmol) was added to theresulting solution and the mixture stirred for 30 mins. The reaction wasquenched by addition of dilute hydrochloric acid (0.1 M), followed byextraction with ethyl acetate (×3). The separated organic phase wassequentially washed with sodium bicarbonate solution and brine beforedrying over sodium sulphate. The volatiles were then removed in vacuo.The residue was purified by flash chromatography on silica eluting withethyl acetate/hexane 1/1. The desired productDiethyl(S)-(E)-3-((tert-butoxycarbonylamino-valyl)alanyl)amino-5-methylhexenylsulfonate,(22 mg, 35%) was obtained as a solid.

[0273] MS(electrospray) required(M⁺(C₂₂H₄₁O₇N₃S)+1)=492: found(M⁺+1)=492, ((M⁺-^(t)Boc)+1)=392 (1000%)

Example 2

[0274] Design of Depsipeptides

[0275] Another suitable bond in a compound of general formula (I), (II)or (III) according to the invention is an ester bond to form adepsipeptide. The incorporation of depsipeptide substrates aided theidentification of substrates for low reactivity viral proteases, such asviral serine proteases.

[0276] For example, substrates of the general formula

n[Abz-B₁₋₁₀-C₁₋₁₀-D₁₋₈ y[COO]-E₁₋₈-Tyr(NO₂)-Asp-NH₂]

[0277] were produced.

[0278] However, a significant proportion of viral proteases onlyrecognise substrate sequences larger than those represented by thegeneral structure above. It is well acknowledged that by the very natureof action of a viral protease (function is to cleave immature viralproteins into the mature viral package) one automatically receives dataconcerning the natural substrate sites. Thus, the general structureabove can be extended by introducing extra fixed amino-acids atappropriate sites. A logical extension would be to introduce the knownP1-P1′ cleavage site as the depsipeptide bond, then subsequentlyintroduce the four variant positions following the standard format thus;

n[Abz-B₁₋₁₀-C₁₋₁₀-D₁₋₈-E₁₋₈-P1 y[COO]-P1′-Tyr(NO₂)-Asp-NH₂]

[0279] Furthermore, if these substrates again proved to be too small,one may use the known substrate sequences to introduce additional fixedpositions. For instance, with Hepatitis NS3 protease it is known thatthe natural P6 position is a conserved acidic residue (aspartic orglutamic acid). Thus one could extend the above structure as detailedbelow.

n[Abz-P6-B₁₋₁₀-C₁₋₁₀-D₁₋₈-E₁₋₁₀-P1 y[COO]-P1′-Tyr(NO₂)-Asp-NH₂]

[0280] The novel methodology described herein greatly faciltiates theinvention of therapeutically useful proteolytic enzyme inhibitors and iscommercially exploitable. This is because the best substrate motif forthe proteolytic enzyme can be rapidly identified, and, since there existin the literature a variety of ways for attaching motifs which reactwith the active site of a proteolytic enzyme, especially for aspartyl,metallo, serine and cysteinyl proteases, an enzyme inhibitor can bereadily synthesised. Moreover, amide bond replacements or transitionstate mimetics can be incorporated into the molecule, which would beespecially useful for the inhibition of aspartyl or metallo proteases.

[0281] The method described also facilitates the rapid development of ascreening assay for novel protease inhibitors. The most potentfluorogenic substrate discovered by library screening can subsequentlybe used for the detection of inhibitors of the particular proteolyticenzyme under scrutiny.

[0282] The presence of an inhibitor within the compound libraries isdescribed is readily detected by retreatment of the assay mixture withthe most active fluorogenic substrate, which will allow the immediatemeasurement of the remaining proteolytic enzyme activity.

[0283] The invention provides self-decoding, combinatorial fluorogeniclibraries, and it will greatly facilitate the design and invention ofnovel protease inhibitors because:

[0284] i. The peptides of the library may have increased aqueoussolubility in comparison to peptides containing similar and otherfluorogenic and quencher groups.

[0285] ii. The peptides are stable to contaminating exopeptidases.

[0286] iii. The self deconvolution-method described, coupled with thecontinuous analysis of the rate of substrate cleavage data, allows theimmediate identification of the most active binding motif containedwithin the substrate library.

[0287] iv. The method allows for the rapid assessment of the enzymeassay mixture for any compounds in the library that are acting as enzymeinhibitors.

[0288] Abbreviations

[0289] Abbreviations used herein are as follows:

[0290] Abbreviations for amino acids and nomenclature of peptidestructures follow the recommendations given in: IUPAC-IUB Commission onBiochemical Nomenclature, (J. Biol. Chem, 247, 997, 1971). All chiralamino acids are of the L configuration unless otherwise stated. Otherabbreviations used are:

[0291] -Abu, b-amino butyric acid,:Abz, 2-amino benzoyl:ACH,1-amino-1-carboxy-cyclohexane:ACP, 1-amino-1-carboxy-cyclopropane:Bip,Biphenylalanine:n-Bu, n-butoxycarbonyl:Bz, Benzoyl:Bz(2-carboxy),2-carboxybenzoyl:Bu^(t)Gly, tert-Butylglycyl:BOP,benzotriazoyl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate:Cha, cyclohexylalanine:Chex,1-carboxycyclohexyl:eAHA, gamma aminohexanoyl:HBTU,O-benzotriazoyl-N,N,N′,N′-tetramethyluronium hexafluorophosphate:HOBt,1-hydroxybenzotriazole:Hyp, trans-4-hydroxyprolinyl:hLeu,homoleucyl:2Nal, 2-napthylalanine:NMM, N-methylmorpholine:Piv, pivoyl3pyr, 3-pyridylalanine:Tic, 2-carboxytetrahydroquinolyl:Tyr(NO₂),3-nitrotyrosine.

[0292] DMF, dimethylformamide; Fmoc, fluorenylmethoxycarbonyl; HPLC,high performance liquid chromatography; Pfp, pentafluorophenyl, tBoc,tert-butoxycarbonyl; tBu, tert-butyl; TFA, trifluoroacetic acid; Pmc,pentamethyl chroman, Pbf, pentamethylbenzofuran, TBTU,2-(1H-Benztrotriazole-1-yl)-1,1,1,3,3-tetramethyluroniumtetrafluoroborate; Trt, Trityl.

[0293] p.Aba, 4-aminobenzoyl; Aib, Aminoisobutyric acid; Bip,Biphenylalanine; nBu, n-Butyl; Bz, Benzoyl; Cmpi,Carboxymethylpiperazine; Deg, Diethylglycine; DIPEA,N,N-Diisopropyl-ethylamine; HATU,0-(7-azabezotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate; HOAT, 1-hydroxy-7-azabenzotriazole; Naph,Naphthylalanine; 3.Pyr, 3-pyridiylalanine; Tyr(NO,), 3-nitro-tyrosine.

REFERENCES

[0294] 1. I. Schlechter and A. Berger, Biochem. Biophys. Res. Commun.,1967, 27, 157-162)

[0295] 2. A. Carmel et al., FEBS Lett., 1973, 30, 11.

[0296] 3 M. M. Meldal and I. Svendsen, J. Chem. Soc. Perkin Trans. 1,1995, 1591-1596,

[0297] 4. M. Meldal and K. Breddam, Anal. Biochem., 1991, 195, 141-147,

[0298] 5. T. Forster, Ann. Phys., 1948, 6, 55.

[0299] 6. A. Yaron, A. Carmel, and E. Katchalski-Katzir, Anal. Biochem.1979, 95, 228 and references therein.

[0300] 7. S. A. Latt et al., Anal. Biochem., 1972, 50, 56.

[0301] 8. A. Persson et al., Anal. Biochem., 1977, 83, 2,96.

[0302] 9. I. Yu Filppova et al., Bioorg. Khim., 1986, 12, 1172.

[0303] 10. J. Pohl et al., Anal. Biochem., 1987, 165, 96.

[0304] 11. S. J. Pollack et al., J. Am. Chem. Soc., 1989, 111, 5961.

[0305] 12. E. K. Bratovanova and D. D. Petkov, Analytical Biochem.,1987, 162, 213.

[0306] 13. J. Singh et al., J. Med. Chem., 1995, 38, 217-219 andreferences. therein.

[0307] 14. M. Green et al. in ‘Innovation and Perspectives in SolidPhase Synthesis’ (R. Epton Ed.) Mayflower Worldwide Ltd., Birmingham,UK. 1994, 239-244.

[0308] 15. J. R. Petithory et al., Proc. Natl. Acad. Sci. USA, 1991, 88,11510-11514

[0309] 16. J. Berman et al., J. Biol. Chem., 1992, 267, 1434-1437.

[0310] 17. H. Drevin, A, -T. Martin, J. Carlsson, S. Oscarsson, T.Lovgren, I. Hemmila and M. Kwiatkowski, WO89/10975, (May 5, 1988)

[0311] 18. A. J. Garman and N. G. Phillips, WO 94/28166 (May 27, 1993)

[0312] 19. G. T. Wang and E. D. Matayoshi, E.P. 428000 (Nov. 3, 1989)

[0313] 20. G. A. Krafft, G. T. Wang and E. D. Matayoshi, EP 428000,(Nov. 3, 1989).

[0314] 21. G. R. Marshall and M. V. Toth, U.S. Pat. No. 5,164,300, (Dec.11, 1990).

[0315] 22. G. R. Marshall and M. V. Toth, U.S. Pat. No. 5,011,910, (Dec.28, 1989).

[0316] 23. K. T. Chapman, N. A. Thornberry, M. Maccoss, J. R. Weidner,R. A. Mumford, W. K. Hagmann, EP 528487A (Aug. 16, 1991)

[0317] 24. R. P. Haugland, WO 93/04077, (Aug. 23, 1991)

[0318] 25. R. M. Valerio, A. M. Bray N. J. Maeji, Int. J. PeptideProtein Res., 1994, 44, 158-165

[0319] 26. M. Bastos, N. J. Maeji and R. H. Abeles, Proc. natl. Acad.Sci., 1995, 92, 6738-6742.

[0320] 27. ‘Solid Phase Peptide Synthesis’, E. Atherton and R. C.Sheppard, IRL Press 1989.

0 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 105 <210> SEQ ID NO 1<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION: Nle <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHERINFORMATION: Tyr(NO2) <400> SEQUENCE: 1 Xaa Val Ala Xaa Ser Xaa Asp 1 5<210> SEQ ID NO 2 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (3) <223> OTHER INFORMATION: Nle <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223> OTHER INFORMATION:Tyr(NO2) <400> SEQUENCE: 2 Val Ala Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 3<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (2) <223> OTHER INFORMATION: Nle <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION: Tyr(NO2)<400> SEQUENCE: 3 Ala Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 4 <211>LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(3) <223> OTHER INFORMATION: Nle <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (5) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 4Val Ala Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 5 <211> LENGTH: 5 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (3) <223> OTHERINFORMATION: Nle <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(5) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 5 Val Ala Xaa SerXaa 1 5 <210> SEQ ID NO 6 <211> LENGTH: 5 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (2) <223> OTHER INFORMATION: Nle<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: Tyr(NO2) <400> SEQUENCE: 6 Ala Xaa Ser Xaa Asp 1 5 <210>SEQ ID NO 7 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (3) <223> OTHER INFORMATION: Nle <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223> OTHER INFORMATION:Tyr(NO2) <400> SEQUENCE: 7 Val Ala Xaa Ser Xaa 1 5 <210> SEQ ID NO 8<211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION: Nle <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHERINFORMATION: Tyr(NO2) <400> SEQUENCE: 8 Xaa Val Ala Xaa Ser Xaa 1 5<210> SEQ ID NO 9 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION: Nle<400> SEQUENCE: 9 Xaa Val Ala Xaa Ser 1 5 <210> SEQ ID NO 10 <211>LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(1) <223> OTHER INFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (4) <223> OTHER INFORMATION: Nle <400> SEQUENCE: 10 XaaVal Ala Xaa Ser Phe Asp 1 5 <210> SEQ ID NO 11 <211> LENGTH: 7 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(4) <223> OTHER INFORMATION: Nle <400> SEQUENCE: 11 Xaa Val Ala Xaa SerTyr Asp 1 5 <210> SEQ ID NO 12 <211> LENGTH: 7 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: Nle <400> SEQUENCE: 12 Xaa Val Ala Xaa Ser Ala Asp 1 5<210> SEQ ID NO 13 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION: Nle<400> SEQUENCE: 13 Xaa Val Ala Xaa Ser Lys Asp 1 5 <210> SEQ ID NO 14<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION: Nle <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHERINFORMATION: eAHA <400> SEQUENCE: 14 Xaa Val Ala Xaa Ser Xaa Asp 1 5<210> SEQ ID NO 15 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (3) <223> OTHER INFORMATION: Nle<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223> OTHERINFORMATION: Tyr(NO2) <400> SEQUENCE: 15 Xaa Ala Xaa Ser Xaa Asp 1 5<210> SEQ ID NO 16 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (2) <223> OTHER INFORMATION: Nle<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: Tyr(NO2) <400> SEQUENCE: 16 Xaa Xaa Ser Xaa Asp 1 5 <210>SEQ ID NO 17 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (3) <223> OTHER INFORMATION: Nle <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223> OTHER INFORMATION:Tyr(NO2) <400> SEQUENCE: 17 Val Ala Xaa Ser Xaa 1 5 <210> SEQ ID NO 18<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (3) <223> OTHER INFORMATION: Nle <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (5) <223> OTHER INFORMATION: Tyr(NO2)<400> SEQUENCE: 18 Val Ala Xaa Ser Xaa 1 5 <210> SEQ ID NO 19 <211>LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(3) <223> OTHER INFORMATION: Nle <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (5) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 19Val Ala Xaa Ser Xaa 1 5 <210> SEQ ID NO 20 <211> LENGTH: 5 <212> TYPE:PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (3) <223> OTHERINFORMATION: Nle <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(5) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 20 Val Ala Xaa SerXaa 1 5 <210> SEQ ID NO 21 <211> LENGTH: 5 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (3) <223> OTHER INFORMATION: Nle<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223> OTHERINFORMATION: Tyr(NO2) <400> SEQUENCE: 21 Val Ala Xaa Ser Xaa 1 5 <210>SEQ ID NO 22 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (3) <223> OTHER INFORMATION: Nle <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223> OTHER INFORMATION:Tyr(NO2) <400> SEQUENCE: 22 Val Ala Xaa Ser Xaa 1 5 <210> SEQ ID NO 23<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2)<400> SEQUENCE: 23 Xaa Val Ala Lys Ser Xaa Asp 1 5 <210> SEQ ID NO 24<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2)<400> SEQUENCE: 24 Xaa Val Ala Gln Ser Xaa Asp 1 5 <210> SEQ ID NO 25<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2)<400> SEQUENCE: 25 Xaa Val Ala Thr Ser Xaa Asp 1 5 <210> SEQ ID NO 26<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION: hLeu<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHERINFORMATION: Tyr(NO2) <400> SEQUENCE: 26 Xaa Val Ala Xaa Ser Xaa Asp 1 5<210> SEQ ID NO 27 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION: Cha<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHERINFORMATION: Tyr(NO2) <400> SEQUENCE: 27 Xaa Val Ala Xaa Ser Xaa Asp 1 5<210> SEQ ID NO 28 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHER INFORMATION:Tyr(NO2) <400> SEQUENCE: 28 Xaa Val Ala His Ser Xaa Asp 1 5 <210> SEQ IDNO 29 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION: ACH<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHERINFORMATION: Tyr(NO2) <400> SEQUENCE: 29 Xaa Val Ala Xaa Ser Xaa Asp 1 5<210> SEQ ID NO 30 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION:DNle <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223>OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 30 Xaa Val Ala Xaa Ser XaaAsp 1 5 <210> SEQ ID NO 31 <211> LENGTH: 7 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: 3pyr <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 31 Xaa Val Ala XaaSer Xaa Asp 1 5 <210> SEQ ID NO 32 <211> LENGTH: 7 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: Hyp <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 32 Xaa Val Ala XaaSer Xaa Asp 1 5 <210> SEQ ID NO 33 <211> LENGTH: 7 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: ACP <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 33 Xaa Val Ala XaaSer Xaa Asp 1 5 <210> SEQ ID NO 34 <211> LENGTH: 7 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 34 Xaa Val Lys XaaSer Xaa Asp 1 5 <210> SEQ ID NO 35 <211> LENGTH: 7 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (3) <223> OTHERINFORMATION: DAla <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(4) <223> OTHER INFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 35Xaa Val Xaa Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 36 <211> LENGTH: 7 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(3) <223> OTHER INFORMATION: Tic <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (4) <223> OTHER INFORMATION: hLeu <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2)<400> SEQUENCE: 36 Xaa Val Xaa Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 37<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (3) <223> OTHER INFORMATION: ACH <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 37 Xaa Val Xaa XaaSer Xaa Asp 1 5 <210> SEQ ID NO 38 <211> LENGTH: 7 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (3) <223> OTHERINFORMATION: Met(O) <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (4) <223> OTHER INFORMATION: hLeu <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2)<400> SEQUENCE: 38 Xaa Val Xaa Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 39<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (3) <223> OTHER INFORMATION: 2Nal<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 39 Xaa Val Xaa XaaSer Xaa Asp 1 5 <210> SEQ ID NO 40 <211> LENGTH: 7 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (3) <223> OTHERINFORMATION: ACP <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(4) <223> OTHER INFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 40Xaa Val Xaa Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 41 <211> LENGTH: 7 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(3) <223> OTHER INFORMATION: DLys <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (4) <223> OTHER INFORMATION: hLeu <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2)<400> SEQUENCE: 41 Xaa Val Xaa Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 42<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (3) <223> OTHER INFORMATION: DGln<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 42 Xaa Val Xaa XaaSer Xaa Asp 1 5 <210> SEQ ID NO 43 <211> LENGTH: 7 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (3) <223> OTHERINFORMATION: 3pyr <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(4) <223> OTHER INFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 43Xaa Val Xaa Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 44 <211> LENGTH: 7 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(3) <223> OTHER INFORMATION: Cha <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (4) <223> OTHER INFORMATION: hLeu <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2)<400> SEQUENCE: 44 Xaa Val Xaa Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 45<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (2) <223> OTHER INFORMATION: DVal<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 45 Xaa Xaa Ala XaaSer Xaa Asp 1 5 <210> SEQ ID NO 46 <211> LENGTH: 7 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 46 Xaa Gln Ala XaaSer Xaa Asp 1 5 <210> SEQ ID NO 47 <211> LENGTH: 7 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 47 Xaa Lys Ala XaaSer Xaa Asp 1 5 <210> SEQ ID NO 48 <211> LENGTH: 7 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (2) <223> OTHERINFORMATION: Tic <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(4) <223> OTHER INFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 48Xaa Xaa Ala Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 49 <211> LENGTH: 7 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(2) <223> OTHER INFORMATION: ACH <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (4) <223> OTHER INFORMATION: hLeu <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2)<400> SEQUENCE: 49 Xaa Xaa Ala Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 50<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (2) <223> OTHER INFORMATION: Met(O)<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 50 Xaa Xaa Ala XaaSer Xaa Asp 1 5 <210> SEQ ID NO 51 <211> LENGTH: 7 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (2) <223> OTHERINFORMATION: 3pyr <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(4) <223> OTHER INFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 51Xaa Xaa Ala Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 52 <211> LENGTH: 7 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(2) <223> OTHER INFORMATION: 2Nal <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (4) <223> OTHER INFORMATION: hLeu <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2)<400> SEQUENCE: 52 Xaa Xaa Ala Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 53<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION: hLeu<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHERINFORMATION: Tyr(NO2) <400> SEQUENCE: 53 Xaa Leu Ala Xaa Ser Xaa Asp 1 5<210> SEQ ID NO 54 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (2) <223> OTHER INFORMATION: Cha<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 54 Xaa Xaa Ala XaaSer Xaa Asp 1 5 <210> SEQ ID NO 55 <211> LENGTH: 7 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (2) <223> OTHERINFORMATION: Bip <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(4) <223> OTHER INFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 55Xaa Xaa Ala Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 56 <211> LENGTH: 7 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(2) <223> OTHER INFORMATION: Bip <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (4) <223> OTHER INFORMATION: hLeu <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2)<400> SEQUENCE: 56 Xaa Xaa Ala Xaa Tyr Xaa Asp 1 5 <210> SEQ ID NO 57<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (2) <223> OTHER INFORMATION: Bip <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 57 Xaa Xaa Ala XaaLeu Xaa Asp 1 5 <210> SEQ ID NO 58 <211> LENGTH: 7 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (2) <223> OTHERINFORMATION: Bip <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(4) <223> OTHER INFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 58Xaa Xaa Ala Xaa Lys Xaa Asp 1 5 <210> SEQ ID NO 59 <211> LENGTH: 7 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(2) <223> OTHER INFORMATION: Bip <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (4) <223> OTHER INFORMATION: hLeu <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2)<400> SEQUENCE: 59 Xaa Xaa Ala Xaa Asp Xaa Asp 1 5 <210> SEQ ID NO 60<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (2) <223> OTHER INFORMATION: Bip <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(5) <223> OTHER INFORMATION: Abu <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 60Xaa Xaa Ala Xaa Xaa Xaa Asp 1 5 <210> SEQ ID NO 61 <211> LENGTH: 7 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(2) <223> OTHER INFORMATION: Bip <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (4) <223> OTHER INFORMATION: hLeu <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (5) <223> OTHER INFORMATION: Cha <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHERINFORMATION: Tyr(NO2) <400> SEQUENCE: 61 Xaa Xaa Ala Xaa Xaa Xaa Asp 1 5<210> SEQ ID NO 62 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (2) <223> OTHER INFORMATION: Bip<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(5) <223> OTHER INFORMATION: Met(O) <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2) <400>SEQUENCE: 62 Xaa Xaa Ala Xaa Xaa Xaa Asp 1 5 <210> SEQ ID NO 63 <211>LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(1) <223> OTHER INFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (2) <223> OTHER INFORMATION: Bip <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION: hLeu<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHERINFORMATION: Tyr(NO2) <400> SEQUENCE: 63 Xaa Xaa Ala Xaa Thr Xaa Asp 1 5<210> SEQ ID NO 64 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (2) <223> OTHER INFORMATION: Bip<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(5) <223> OTHER INFORMATION: 3pyr <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 64Xaa Xaa Ala Xaa Xaa Xaa Asp 1 5 <210> SEQ ID NO 65 <211> LENGTH: 7 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(2) <223> OTHER INFORMATION: Bip <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (4) <223> OTHER INFORMATION: hLeu <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (5) <223> OTHER INFORMATION: BuGly<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHERINFORMATION: Tyr(NO2) <400> SEQUENCE: 65 Xaa Xaa Ala Xaa Xaa Xaa Asp 1 5<210> SEQ ID NO 66 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (2) <223> OTHER INFORMATION: Bip<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: hLeu <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(5) <223> OTHER INFORMATION: Hyp <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 66Xaa Xaa Ala Xaa Xaa Xaa Asp 1 5 <210> SEQ ID NO 67 <211> LENGTH: 8 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(5) <223> OTHER INFORMATION: Nle <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (7) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 67Xaa Phe Val Ala Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 68 <211> LENGTH: 8<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(2) <223> OTHER INFORMATION: 3.Pyr <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (5) <223> OTHER INFORMATION: Nle <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (7) <223> OTHER INFORMATION:Tyr(NO2) <400> SEQUENCE: 68 Xaa Xaa Val Ala Xaa Ser Xaa Asp 1 5 <210>SEQ ID NO 69 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (2) <223> OTHER INFORMATION:1.Naph <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223>OTHER INFORMATION: Nle <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (7) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 69 XaaXaa Val Ala Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 70 <211> LENGTH: 8 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(2) <223> OTHER INFORMATION: 2.Naph <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (5) <223> OTHER INFORMATION: Nle <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (7) <223> OTHER INFORMATION:Tyr(NO2) <400> SEQUENCE: 70 Xaa Xaa Val Ala Xaa Ser Xaa Asp 1 5 <210>SEQ ID NO 71 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223> OTHER INFORMATION: Nle<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (7) <223> OTHERINFORMATION: Tyr(NO2) <400> SEQUENCE: 71 Xaa Tyr Val Ala Xaa Ser Xaa Asp1 5 <210> SEQ ID NO 72 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (2) <223> OTHER INFORMATION: Bip<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223> OTHERINFORMATION: Nle <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(7) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 72 Xaa Xaa Val AlaXaa Ser Xaa Asp 1 5 <210> SEQ ID NO 73 <211> LENGTH: 8 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(5) <223> OTHER INFORMATION: Nle <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (7) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 73Xaa Lys Val Ala Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 74 <211> LENGTH: 8<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(5) <223> OTHER INFORMATION: Nle <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (7) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 74Xaa Glu Val Ala Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 75 <211> LENGTH: 8<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(5) <223> OTHER INFORMATION: Nle <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (7) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 75Xaa Leu Val Ala Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 76 <211> LENGTH: 8<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(2) <223> OTHER INFORMATION: Hyp <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (5) <223> OTHER INFORMATION: Nle <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (7) <223> OTHER INFORMATION: Tyr(NO2)<400> SEQUENCE: 76 Xaa Xaa Val Ala Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO77 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION: (NMe)Nle<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHERINFORMATION: Tyr(NO2) <400> SEQUENCE: 77 Xaa Val Ala Xaa Ser Xaa Asp 1 5<210> SEQ ID NO 78 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (3) <223> OTHER INFORMATION:(NMe)Ala <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4)<223> OTHER INFORMATION: Nle <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 78Xaa Val Xaa Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 79 <211> LENGTH: 7 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(4) <223> OTHER INFORMATION: Aib <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 79Xaa Val Ala Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 80 <211> LENGTH: 7 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(3) <223> OTHER INFORMATION: Aib <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (4) <223> OTHER INFORMATION: Nle <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (6) <223> OTHER INFORMATION: Tyr(NO2)<400> SEQUENCE: 80 Xaa Val Xaa Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 81<211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (2) <223> OTHER INFORMATION: Deg <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: Nle <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(6) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 81 Xaa Xaa Ala XaaSer Xaa Asp 1 5 <210> SEQ ID NO 82 <211> LENGTH: 6 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: nBu<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (2) <223> OTHERINFORMATION: D.Ser <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (3) <223> OTHER INFORMATION: D.Nle <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION: D.Ala<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223> OTHERINFORMATION: D.Val <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (6) <223> OTHER INFORMATION: p.Aba <400> SEQUENCE: 82 Xaa XaaXaa Xaa Xaa Xaa 1 5 <210> SEQ ID NO 83 <211> LENGTH: 6 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Bz <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(4) <223> OTHER INFORMATION: Statine <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (6) <223> OTHER INFORMATION: eAha <400>SEQUENCE: 83 Xaa Val Ala Xaa Ser Xaa 1 5 <210> SEQ ID NO 84 <211>LENGTH: 6 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(1) <223> OTHER INFORMATION: Abz <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (2) <223> OTHER INFORMATION: p.Aba <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (3) <223> OTHER INFORMATION: Nle <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223> OTHERINFORMATION: Tyr(NO2) <400> SEQUENCE: 84 Xaa Xaa Xaa Ser Xaa Asp 1 5<210> SEQ ID NO 85 <211> LENGTH: 6 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: Abz <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (2) <223> OTHER INFORMATION:Cmpi <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (3) <223>OTHER INFORMATION: Nle <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (5) <223> OTHER INFORMATION: Tyr(NO2) <400> SEQUENCE: 85 XaaXaa Xaa Ser Xaa Asp 1 5 <210> SEQ ID NO 86 <211> LENGTH: 4 <212> TYPE:PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: N-Benzoyl <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (4) <223> OTHER INFORMATION: L-norleucine <400> SEQUENCE: 86Xaa Val Ala Xaa 1 <210> SEQ ID NO 87 <211> LENGTH: 4 <212> TYPE: PRT<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: N-Benzoyl <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (4) <223> OTHER INFORMATION: L-norleucine bromomethyl ketone<400> SEQUENCE: 87 Xaa Val Ala Xaa 1 <210> SEQ ID NO 88 <211> LENGTH: 4<212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223>OTHER INFORMATION: Description of Artificial Sequence: Synthetic peptide<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: N-Benzoyl <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (4) <223> OTHER INFORMATION: L-norleucine 2, 6-bis(trifluoromethyl) benzoyloxymethyl ketone <400> SEQUENCE: 88 Xaa Val AlaXaa 1 <210> SEQ ID NO 89 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: N-Benzoyl <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: L-norleucine 2, 6-dimethylbenzoyloxymethyl ketone <400>SEQUENCE: 89 Xaa Val Ala Xaa 1 <210> SEQ ID NO 90 <211> LENGTH: 4 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: N-Benzoyl <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (4) <223> OTHER INFORMATION: L-norleucine2-hydroxybenzoyloxymethyl ketone <400> SEQUENCE: 90 Xaa Val Ala Xaa 1<210> SEQ ID NO 91 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM:Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION: Descriptionof Artificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION: N-Benzoyl <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: L-norleucine 2, 6-dichlorobenzoyloxymethyl ketone <400>SEQUENCE: 91 Xaa Val Ala Xaa 1 <210> SEQ ID NO 92 <211> LENGTH: 4 <212>TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: N-Benzoyl <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (4) <223> OTHER INFORMATION: L-norleucine benzoyloxymethylketone <400> SEQUENCE: 92 Xaa Val Ala Xaa 1 <210> SEQ ID NO 93 <211>LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(1) <223> OTHER INFORMATION: N-Benzoyl <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION: L-norleucine 2, 3,4, 5, 6-pentafluorobenzoyloxymethyl ketone <400> SEQUENCE: 93 Xaa ValAla Xaa 1 <210> SEQ ID NO 94 <211> LENGTH: 4 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION:N-Benzoyl <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4)<223> OTHER INFORMATION: L-norleucine 1, 1-dimethylpropyloxymethylketone <400> SEQUENCE: 94 Xaa Val Ala Xaa 1 <210> SEQ ID NO 95 <211>LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(1) <223> OTHER INFORMATION: N-Benzoyl <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION: L-norleucine <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223> OTHERINFORMATION: N(-benxyloxycarbonyl)-D-serinyl -(0-tert-butyl) oxymethylketone <400> SEQUENCE: 95 Xaa Val Ala Xaa Xaa 1 5 <210> SEQ ID NO 96<211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence<220> FEATURE: <223> OTHER INFORMATION: Description of ArtificialSequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222>LOCATION: (1) <223> OTHER INFORMATION: N-Benzoyl <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION:L-norleucine <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (5)<223> OTHER INFORMATION: N(-benxyloxycarbonyl)-D-serineoxy methyl ketone<400> SEQUENCE: 96 Xaa Val Ala Xaa Xaa 1 5 <210> SEQ ID NO 97 <211>LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(1) <223> OTHER INFORMATION: N-Benzoyl <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION: L-norleucine <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (5) <223> OTHERINFORMATION: 2-furanoxy methyl ketone <400> SEQUENCE: 97 Xaa Val Ala XaaXaa 1 5 <210> SEQ ID NO 98 <211> LENGTH: 4 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION:N-Benzoyl <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4)<223> OTHER INFORMATION: L-norleucine 2, 6-dichlorophenylacyloxy methylketone <400> SEQUENCE: 98 Xaa Val Ala Xaa 1 <210> SEQ ID NO 99 <211>LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(1) <223> OTHER INFORMATION: Ethyl-(S)-(E)-3-(tert-butoxy carbonyl)<220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHERINFORMATION: amino-6-methyl-hept-2-enoate <400> SEQUENCE: 99 Xaa Val AlaXaa 1 <210> SEQ ID NO 100 <211> LENGTH: 4 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION:(S)-(E)-3-(tert-butoxy carbonyl) <220> FEATURE: <221> NAME/KEY: MOD_RES<222> LOCATION: (4) <223> OTHER INFORMATION: amino-6-methyl-hept-2-enoicacid <400> SEQUENCE: 100 Xaa Val Ala Xaa 1 <210> SEQ ID NO 101 <211>LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220>FEATURE: <223> OTHER INFORMATION: Description of Artificial Sequence:Synthetic peptide <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION:(1) <223> OTHER INFORMATION: 1,1,1-Trifluoroethyl-(S)-(E)-3-(tert-butoxycarbonyl) <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4)<223> OTHER INFORMATION: amino-6-methyl-hept-2-enoate <400> SEQUENCE:101 Xaa Val Ala Xaa 1 <210> SEQ ID NO 102 <211> LENGTH: 4 <212> TYPE:PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Description of Artificial Sequence: Synthetic peptide <220>FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHERINFORMATION: Ethyl-(S)-(E)-3-(N-benzoyl) <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION:amino-6-methyl-hept-2-enoate <400> SEQUENCE: 102 Xaa Val Ala Xaa 1 <210>SEQ ID NO 103 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION:(S)-(E)-3-(tert-butoxycarbonylamino) <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION:amino-1-phenylsulfonyl-5-methyl-1-hexene <400> SEQUENCE: 103 Xaa Val AlaXaa 1 <210> SEQ ID NO 104 <211> LENGTH: 4 <212> TYPE: PRT <213>ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHER INFORMATION:Description of Artificial Sequence: Synthetic peptide <220> FEATURE:<221> NAME/KEY: MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION:(S)-(E)-3-(tert-butoxycarbonylamino) <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION:amino-1-methylsulfonyl-5-methyl-1-hexene <400> SEQUENCE: 104 Xaa Val AlaXaa 1 SEQ ID NO 105 LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: ArtificialSequence <220> FEATURE: <223> OTHER INFORMATION: Description ofArtificial Sequence: Synthetic peptide <220> FEATURE: <221> NAME/KEY:MOD_RES <222> LOCATION: (1) <223> OTHER INFORMATION:Diethyl(S)-(E)-3-(tert-butoxycarbonylamino) <220> FEATURE: <221>NAME/KEY: MOD_RES <222> LOCATION: (4) <223> OTHER INFORMATION:amino-5-methylhexenylsulfonate <400> SEQUENCE: 105 Xaa Val Ala Xaa 1

1. A complementary pair of compound libraries L1 and L2 which constitutea set containing combinatorial FRET compounds of formula:Aa-Bb-Cc-Dd-n(Ee)-Ff-Gg in which; A represents a fluorescor internallyquenched by F; B, C, D, and E represent groups such that the scissilebond between any two of these groups is a suitable bond; F represents aquencher capable of internally quenching the fluorescor A; and whereinall compounds are in aqueous solution; and n represents an intergerbetween 1 and 4 inclusive; giving a×b×c×d×e×f×g=Mn compounds in eachlibrary, there being a predetermined number (P1, P2) of mixtures eachconsisting of a predetermined number (Q1, Q2) of individual identifiablecompounds in each library, wherein both L1 and L2 contain the same Mncompounds, but wherein any two compounds which are found together in onemixture of Q1 compounds of L1 are not found together in any one of theP2 mixtures of L2.
 2. A method of screening for enzymic activity,wherein the activity is the interaction of proteolytic enzyme applied toa well with one or more compounds of a mixture in the well, using thelibraries L1, L2 according to claim 1, in which the P1 mixtures of L1and the P2 mixtures of L2 are each placed separately into individualwells of well plates, the well plates having wells arranged in a formatadapted to allow deduction of a unique active compound formula from thepresence of activity in one well of L1 and one well of L2.
 3. A methodaccording to claim 2 wherein the format complies with generaldeconvolution formulae in which:${(i)\quad n\quad s} = {\frac{{Rp} \cdot {Cp} \cdot}{{Rs} \cdot {Cs}}N\quad p}$

(ii) k=b.c.d.np.e (iii) k=x.N.np (iv) N=Rp.Cp (v) K=X.Rp.Cp.np (vi)b.c.d.e=X.Rp.Cp (vii) Cp.e=X (viii) Rp.e=X, if Rp=Cp and whereinnp=number of primary plates ns=number of secondary plates Rp=number ofprimary rows Rs=number of secondary rows Cp=number of primary columnsCs=number of secondary columns K=number of combinations of compoundsN=number of wells on a plate, and x=number of compounds per well.
 4. Amethod according to claim 3 wherein n=4 ns=16 Rp=8 Rs=4 Cp=10 Cs=5K=6400 N=80 x=20
 5. A library pair according to claim 1 wherein thescissile bond is between D and E.
 6. A library pair according to claim 1wherein A represents an unsubstituted or substituted anthranilic acidderivative.
 7. A library pair according to claim 1 wherein B, C, D and Eindependently represent natural or unnatural amino acid residues.
 8. Alibrary pair according to claim 1 wherein F represents an unsubstitutedor substituted 3-nitrotyrosine derivative.
 9. A library pair accordingto claim 1 wherein the scissile bond is a suitable bond selected fromthe group consisting of an unsubstituted amide bond, and an ester bond.10. A library pair according to claim 1 wherein the formula furthercomprises G which ensures the compound is imparted with aqueoussolubility.
 11. A library pair according to claim 10 which isrepresented by the formula A-B-C-D-n(E)-F-G in which A, B, C, D, E, Fand n are as defined in claim 1; and G represents a hydrophilic moietywhich is not an enzyme substrate.
 12. A library pair according to claim10 or 11 of general formula: Abz-B-C-D-E-Tyr(NO2)-Asp-NH2 WhereB=Valine>Alanine, Glutamine, Leucine, PhenylalanineC=Alanine>>Glutamine, or Lysine. D=Leucine, Norleucine or Alanine>SerineE=Serine
 13. Apparatus which comprises an auto-deconvoluting set ofcompounds according to claim 1 that facilitates the invention of novelinhibitors of proteolytic enzymes and the rapid generation ofstructure-activity relationships (SAR) by the detection and measurementof proteolytic enzyme activity.
 14. Apparatus according to claims 13wherein each library comprises a mixture of compounds represented by theformula: A-B₁₋₁₀-C₁₋₁₀-D₁₋₈-n (E₁₋₂)-F.
 15. Apparatus according to claim14 wherein the formula further comprises G which ensures that compoundsin the library are imparted with aqueous solubility.
 16. Apparatusaccording to claim 15 wherein each library comprises a mixture ofcompounds represented by the formula: A-B₁₋₁₀-C₁₋₁₀-D₁₋₈-n (E₁₋₂)-F-G inwhich; G represents a hydrophilic moiety which is not an enzymesubstrate.
 17. Apparatus according to claim 15 or 16 in which Grepresents an aspartyl amide moiety.
 18. Apparatus according to any oneof claims 13 to 17 wherein the library comprises 1600n compounds as 80nmixtures of 20 distinct, identifiable compounds.
 19. Apparatus accordingto claim 18 wherein the mixtures of 20 distinct, identifiable compoundsare placed separately into each of 80 wells of an microtitre plate. 20.Use of a combinatorial FRET library pair according to claim 1 in amethod which provides rapid generation of structure-activityrelationships (SAR) which comprises detection and measurement ofproteolytic enzyme activity by carrying out an assay with a library pairof combinatorial FRET (fluorescence resonance energy transfer) moleculesto find a substrate or substrates for the enzyme.
 21. Use of acombinatorial FRET library pair according to claim 1 in a method fordetection and measurement of proteolytic enzyme activity againstcompounds of the library.
 22. A method of identifying and synthesisingan inhibitor of a proteolytic enzyme which comprises detection andmeasurement of proteolytic enzyme activity by carrying out an assay witha library pair of combinatorial FRET (fluorescence resonance energytransfer) molecules according to claim 1, deconvoluting the library tofind a substrate or substrates for the enzyme and synthesis of aninhibitor based on the substrate or substrates.
 23. An inhibition assaywhich uses a FRET molecule, which has been identified as a substrate forthe enzyme by the method of claim 2, wherein the molecule is assayedwith the enzyme separately against a panel of possible inhibitors.
 24. Amethod which provides the rapid generation of structure-activityrelationships (SAR) which comprises detection and measurement ofproteolytic enzyme activity by carrying out an assay with a library pairof combinatorial FRET (fluorescence resonance energy transfer) moleculesaccording to claim 1 to find a substrate or substrates for the enzyme.25. A method which comprises the identification of an enzyme inhibitoror inhibitors wherein a FRET compound which has been identified as asubstrate by the method of claim 2 is used in an inhibition assay withthe enzyme separately against a panel of possible inhibitors.
 26. Amethod according to claim 25 wherein the assay is performed by using theapparatus according to any of claims 13 to
 19. 27. A method according toany preceding method claim wherein the compounds of the library pair aresynthesized using a solid phase technique.
 28. A library pair,apparatus, method or assay substantially as described herein withreference to example 1.